Pyrimidine compounds as jak kinase inhibitors

ABSTRACT

The invention provides compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     wherein the variables are defined in the specification, or a pharmaceutically-acceptable salt thereof, that are inhibitors of JAK kinases. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat gastrointestinal and other inflammatory diseases, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/328,737, filed on Apr. 28, 2016; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to pyrimidine compounds useful as JAK kinaseinhibitors. The invention is also directed to pharmaceuticalcompositions comprising such compounds, methods of using such compoundsto treat inflammatory diseases, and processes and intermediates usefulfor preparing such compounds.

State of the Art

Ulcerative colitis is a chronic inflammatory disease of the colon. Thedisease is characterized by inflammation and ulceration of the mucosallayer of the rectum and the large intestine. Common symptoms includediarrhea, bloody stools, and abdominal pain. The clinical course isintermittent, marked by alternating periods of exacerbation andremission. Incidence seems to be greater in developed than in developingcountries. An estimated 1.2 million people in major industrializedcountries suffer from ulcerative colitis and the numbers are expected toincrease along with population growth. Patients with ulcerative colitisare at an increased risk of developing colorectal cancer. (e.g. Daneseet al. N Engl J Med, 2011, 365, 1713-1725).

Although there exists a variety of therapeutic options to promote andmaintain remission of ulcerative colitis (UC) in patients, none isideal. Sulfasalazine-related treatments are often effective in mild UC,but much less so in moderate to severe disease. Corticosteroids areoften used to provide rapid induction of remission in patients withmoderate to severe UC. However, chronic use of steroids to maintainremission is discouraged due to their association with longer termadverse effects (e.g., osteoporosis and fractures, infections,cataracts, slower wound healing and suppression of adrenal gland hormoneproduction). Systemic immunosuppressants such as azathioprine,cyclosporine and methotrexate have a slow onset and modest efficacy inmoderate to severe UC patients, but prolonged use can be problematic dueto consequences of long-term systemic immunosuppression (e.g., increasedrisk of infections and lymphoma). Anti-TNFα antibodies (e.g., infliximaband adalimumab), while expensive and requiring subcutaneous orintravenous administration, are efficacious in approximately 60 to 70%of UC patients with moderate to severe disease. However, up to one thirdof patients fail to respond adequately, while another third of initialresponders develop tolerance over a few weeks (Allez et al., J Crohn'sColitis, 2010, 4, 355-366; Rutgeerts et al., N Engl J Med, 2005, 353,2462-2476). The most recently approved UC therapy, vedolizumab, ananti-integrin α₄β₇ antibody, is efficacious in moderate to severe UCpatients although its parenteral route is suboptimal, and theconsequences of long-term immunosuppression via this mechanism remain tobe determined. Despite existing therapeutic options, about 10 to 20% ofUC patients still require colectomy within 10 years of diagnosis(Targownik et al., Am J Gastroenterol, 2012, 107, 1228-1235). It isclear there remains an unmet medical need for an effective therapy topromote and maintain remission of moderate to severe UC without thesafety concerns resulting from chronic, systemic immunosuppression.

While the mechanism underlying ulcerative colitis is not completelyunderstood, it is believed that environmental factors in geneticallysusceptible individuals evoke an inappropriate (excessive) reaction bythe immune system to gut microbiota, resulting in colonic inflammation,tissue damage, and the associated symptoms characteristic of thedisease.

Although the precise pathogenesis of UC is unclear, it is apparent thatproinflammatory cytokines play a pivotal role in the immunologicalresponse (Strober et al., Gastroenterol, 2011, 140, 1756-1767). Many ofthe proinflammatory cytokines most commonly elevated in UC (e.g., IL-4,IL-6, IL-13, IL-15, IL-23, IL-24, IFNγ and leptin), rely on the JAKfamily of tyrosine kinases (i.e., JAK1, JAK2, JAK3 and Tyk2) for signaltransduction. Binding of a cytokine to a JAK-dependent cytokine receptorinduces receptor dimerization which results in phosphorylation oftyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings.

Atopic dermatitis (AD) is a common chronic inflammatory skin diseasethat affects an estimated 14 million people in the United States alone.It is estimated that AD affects 10 to 20% of children and 1 to 3% ofadults in developed countries (Bao et al., JAK-STAT, 2013, 2, e24137)and the prevalence is increasing. Elevation of proinflammatory cytokinesthat rely on the JAK-STAT pathway, in particular, IL-4, IL-5, IL-10,IL-12, IL-13, IFNγ, and TSLP has been associated with AD (Bao et al.,Leung et al., The Journal of Clinical Investigation, 2004, 113,651-657). In addition, upregulation of IL-31, another cytokine thatsignals through a JAK pairing, has been shown to have a role in thepruritis associated with the chronic state of AD (Sunkoly et al.,Journal of Allergy and Clinical Immunology, 2006, 117, 411-417).

Inhibition of the family of JAK enzymes could inhibit signaling of manykey pro-inflammatory cytokines. Thus JAK inhibitors are likely to beuseful in the treatment of ulcerative colitis, and othergastrointestinal inflammatory diseases such as Crohn's disease andimmune checkpoint inhibitor induced colitis, atopic dermatitis, andother inflammatory skin diseases, allergic rhinitis, asthma, and chronicobstructive pulmonary disease (COPD). However, due to the modulatingeffect of the JAK/STAT pathway on the immune system, systemic exposureto JAK inhibitors may have an adverse systemic immunosuppressive effect.It would be desirable, therefore, to provide new JAK inhibitors whichhave their effect at the site of action without significant systemiceffects. In particular, for the treatment of gastrointestinalinflammatory diseases, such as ulcerative colitis, it would be desirableto provide new JAK inhibitors which can be administered orally andachieve therapeutically relevant exposure in the gastrointestinal tractwith minimal systemic exposure. It would also be desirable to providenew JAK inhibitors for the treatment of atopic dermatitis, which can beadministered topically with minimal systemic exposure.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors.

Accordingly, the invention provides a compound of formula (I):

wherein

-   -   R¹ is selected from:        -   (a) —S(O)₂R⁴, wherein R⁴ is selected from:            -   C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted                with —CN, —OC₁₋₃alkyl, or C₃₋₆cycloalkyl,            -   heterocyclyl containing 4 to 6 ring atoms including one                nitrogen atom,                -   wherein any heterocyclyl is optionally substituted                    with —CN, C₃₋₆cycloalkyl,            -   pyridinyl, wherein pyridinyl is optionally substituted                with fluoro, and phenyl;    -   (b) C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with        —CN,

-   -   or        -   pyridinyl, wherein pyridinyl is optionally substituted with            —CN; and    -   (c) —C(O)R⁵, wherein R⁵ is selected from:        -   C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with            C₃₋₆cycloalkyl, or with one or two fluoro,        -   —OC₁₋₄alkyl,        -   C₃₋₆cycloalkyl, and        -   morpholinyl;

R² is hydrogen or methyl;

R³ is C₁₋₃alkyl; and

n is 1 or 2;

or a pharmaceutically-acceptable salt or stereoisomer thereof.

As used hereinafter, the phrase “compound of formula (I)” means acompound of formula (I) or a pharmaceutically acceptable salt thereof;i.e., this phrase means a compound of formula (I) in free base form orin a pharmaceutically acceptable salt form unless otherwise indicated.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

In another aspect, the invention provides a particular compound offormula (I) in crystalline free base form. Crystalline1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrilehas been found to have a melting temperature in the range of about 235°C. to about 245° C., typically between about 237° C. and about 242° C.,and to exhibit weight changes of less than about 0.4% when exposed to arange of relative humidity between about 5% and about 90% at roomtemperature.

The invention also provides a method of treating gastrointestinalinflammatory disease, in particular, ulcerative colitis, in a mammal,the method comprising administering to the mammal a compound or apharmaceutical composition of the invention.

In yet another method aspect, the invention provides a method oftreating inflammatory diseases or disorders of the skin, in particularatopic dermatitis, in a mammal, the method comprising applying acompound or a pharmaceutical composition of the invention to the skin ofthe mammal.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating gastrointestinal inflammatory disease in a mammal. Theinvention further provides the use of a compound of the invention in themanufacture of a formulation or medicament for treating inflammatorydiseases of the skin.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of crystallineForm I1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile[hereinafter Form I].

FIG. 2 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form I.

FIG. 3 shows a thermal gravimetric analysis (TGA) plot of crystallineForm I.

FIG. 4 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm I observed at a temperature of about 25° C.

FIG. 5 shows a powder X-ray diffraction (PXRD) pattern of crystallineForm II1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile[hereinafter Form II].

FIG. 6 shows a differential scanning calorimetry (DSC) thermogram ofcrystalline Form II.

FIG. 7 shows a thermal gravimetric analysis (TGA) plot of crystallineForm II.

FIG. 8 shows a dynamic moisture sorption (DMS) isotherm of crystallineForm II observed at a temperature of about 25° C.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides JAK kinase inhibitors offormula (I), pharmaceutically-acceptable salts thereof, andintermediates for the preparation thereof. The following substituentsand values are intended to provide representative examples of variousaspects of this invention. These representative values are intended tofurther define such aspects and are not intended to exclude other valuesor limit the scope of the invention.

In one aspect of the invention, R¹ is selected from (a) —S(O)₂R⁴,wherein R⁴ is selected from C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with —CN, —OC₁₋₃alkyl, or C₃₋₆cycloalkyl; heterocyclylcontaining 4 to 6 ring atoms including one nitrogen atom, wherein anyheterocyclyl is optionally substituted with —CN; C₃₋₆cycloalkyl;pyridinyl, wherein pyridinyl is optionally substituted with fluoro; andphenyl; (b) C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with—CN,

or pyridinyl, wherein pyridinyl is optionally substituted with —CN; and(c) C(O)R⁵, wherein R⁵ is selected from C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with C₃₋₆cycloalkyl, or with one or two fluoro;—OC₁₋₄alkyl; C₃₋₆cycloalkyl; and morpholinyl.

In another aspect R¹ is —S(O)₂R⁴, wherein R⁴ is selected from C₁₋₄alkyl,wherein C₁₋₄alkyl is optionally substituted with —CN, —OC₁₋₃alkyl, orC₃₋₆cycloalkyl; heterocyclyl containing 4 to 6 ring atoms including onenitrogen atom wherein any heterocyclyl is optionally substituted with—CN; C₃₋₆cycloalkyl; pyridinyl, wherein pyridinyl is optionallysubstituted with fluoro; and phenyl.

In yet another aspect, R¹ is —S(O)₂R⁴, wherein R⁴ is selected fromC₁₋₂alkyl, wherein C₁₋₂alkyl is optionally substituted with —CN, —OCH₃,or cyclopropyl; azetidinyl, wherein azetidinyl is optionally substitutedwith —CN; pyrrolidinyl; cyclopropyl; pyridinyl, wherein pyridinyl isoptionally substituted with fluoro; and phenyl.

In still another aspect, R¹ is —S(O)₂R⁴, wherein R⁴ is methyl, ethyl,azetidinyl, pyrrolidinyl, cyclopropyl, pyridinyl, or phenyl, whereinethyl is optionally substituted with methoxy, azetidinyl is optionallysubstituted with —CN, and pyridinyl is optionally substituted withfluoro.

In one aspect, R¹ is C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with —CN,

or pyridinyl, wherein pyridinyl is optionally substituted with —CN.

In another aspect, R¹ is C₁₋₂alkyl, wherein C₁₋₂alkyl is optionallysubstituted with —CN,

or pyridinyl, wherein pyridinyl is optionally substituted with —CN.

In one aspect, R¹ is —C(O)R⁵, wherein R⁵ is selected from C₁₋₄alkyl,wherein C₁₋₄alkyl is optionally substituted with C₃₋₆cycloalkyl, or withone or two fluoro; —OC₁₋₄alkyl; C₃₋₆cycloalkyl; and morpholinyl.

In another aspect, R¹ is —C(O)R⁵, wherein R⁵ is selected from C₁₋₂alkyl,wherein C₁₋₂alkyl is optionally substituted with cyclopropyl, or withone or two fluoro; —OC₁₋₄alkyl; C₃₋₆cycloalkyl; and morpholinyl.

In yet another aspect, R¹ is —C(O)R⁵, wherein R⁵ is —CHF₂,—CH₂-cyclopropyl, —OCH₃, —O-isobutyl, cyclobutyl, cyclopentyl, ormorpholinyl.

In one aspect R² is hydrogen or methyl. In a specific aspect R² ismethyl.

In one aspect, R³ is C₁₋₃alkyl.

In another aspect, R³ is methyl.

In one aspect, n is 1 or 2. In another aspect, n is 2.

In a certain aspect, the invention provides compounds of formula (II):

wherein the variable R⁴ is as defined herein.

In one aspect, the invention provides the compounds of Examples 1-9 andTables 1-3 below.

In another aspect, the invention provides a compound selected from:

-   1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)sulfonyl)azetidine-3-carbonitrile,-   1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,-   (3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(methylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((6-methoxy-2-(((1R,3s,5S)-9-((2-methoxyethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   3-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile,-   5-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,-   5-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)nicotinonitrile,-   isobutyl    (1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,-   2,2-difluoro-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,-   (3-((2-(((1R,3s,5S)-9-(azetidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,-   (3-((2-(((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((6-methoxy-2-(((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-(ethylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-((cyclopropylmethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((6-methoxy-2-(((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   3-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)-sulfonyl)propanenitrile,-   (3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyrrolidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-(cyclopropylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-(azetidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-((cyclopropylmethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   (3-((2-(((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   4-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,-   (3-((6-methoxy-2-(((1R,3s,5S)-9-(pyridin-3-ylmethyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   3-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)propanenitrile,-   1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)cyclopropane-1-carbonitrile,-   (3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyridin-4-ylmethyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,-   4-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,-   2,2-difluoro-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,-   isobutyl    (1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,-   methyl    (1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,-   ((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxy-pyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)(morpholino)methanone,-   2-cyclopropyl-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,-   cyclopentyl((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methanone,    and-   cyclobutyl((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methanone,-   and pharmaceutically acceptable salts thereof.

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).For example, the compound of Example 2:

is designated as1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile.The (1R,3s,5S) notation describes the exo orientation of thepyrimidinylamino group with respect to the 9-azabicyclo[3.3.1]nonanegroup and similarly for the compounds containing an8-azabicyclo[3.2.1]group (i.e. variable n=1). All of the compounds ofthe invention are in the exo orientation.

Furthermore, the pyrazolyl moiety of the compounds of formula (I) existsin tautomeric form. For example, the compound of Example 2 mayequivalently be represented as:

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the pyrazolyl portion. The aboverepresentation is designated1-(((1R,3s,5S)-3-((4-((3-(hydroxymethyl)-1H-pyrazol-5-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,where the underlining identifies where the name differs from that of thefirst representation. It will be understood that although structures areshown, or named, in a particular form, the invention also includes thetautomer thereof.

The compounds of the invention contain one or more chiral centers andtherefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Compounds of formula (I) also contain several basic groups (e.g., aminogroups) and therefore, such compounds can exist as the free base or invarious salt forms, such a mono-protonated salt form, a di-protonatedsalt form, a tri-protonated salt form, or mixtures thereof. All suchforms are included within the scope of this invention, unless otherwiseindicated.

This invention also includes isotopically-labeled compounds of formula(I), i.e., compounds of formula (I) where an atom has been replaced orenriched with an atom having the same atomic number but an atomic massdifferent from the atomic mass that predominates in nature. Examples ofisotopes that may be incorporated into a compound of formula (I)include, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³⁵S, and ¹⁸F. Of particular interest are compounds of formula(I) enriched in tritium or carbon-14, which compounds can be used, forexample, in tissue distribution studies. Also of particular interest arecompounds of formula (I) enriched in deuterium especially at a site ofmetabolism, which compounds are expected to have greater metabolicstability. Additionally of particular interest are compounds of formula(I) enriched in a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, which compounds can be used, for example, in Positron EmissionTomography (PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl (Me),ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl(n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu),n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “alkoxy” means the monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example, cyclopropyl(cPr), cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,adamantyl, and the like.

The term “heterocyclyl”, “heterocycle”, “heterocyclic”, or “heterocyclicring” means a monovalent saturated or partially unsaturated cyclicnon-aromatic group, having from 3 to 10 total ring atoms, wherein thering contains from 2 to 9 carbon ring atoms and from 1 to 4 ringheteroatoms selected from nitrogen, oxygen, and sulfur. Heterocyclicgroups may be monocyclic or multicyclic (i.e., fused or bridged).Representative heterocyclic groups include, by way of example,pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, morpholinyl,thiomorpholyl, indolin-3-yl, 2-imidazolinyl, tetrahydropyranyl,1,2,3,4-tetrahydroisoquinolin-2-yl, quinuclidinyl, 7-azanorbornanyl,nortropanyl, and the like, where the point of attachment is at anyavailable carbon or nitrogen ring atom. Where the context makes thepoint of attachment of the heterocyclic group evident, such groups mayalternatively be referred to as a non-valent species, i.e. pyrrolidine,piperidine, piperazine, imidazole, tetrahydropyran etc.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition (such as a gastrointestinal inflammatorydisease), in a patient, such as a mammal (particularly a human) whichincludes one or more of the following:

(a) preventing the disease, disorder, or medical condition fromoccurring, i.e., preventing the reoccurrence of the disease or medicalcondition or prophylactic treatment of a patient that is pre-disposed tothe disease or medical condition;

(b) ameliorating the disease, disorder, or medical condition, i.e.,eliminating or causing regression of the disease, disorder, or medicalcondition in a patient, including counteracting the effects of othertherapeutic agents;

(c) suppressing the disease, disorder, or medical condition, i.e.,slowing or arresting the development of the disease, disorder, ormedical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medicalcondition in a patient.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula (I), i.e. a form where one or more amino groupshave been protonated by an acid. Typically, the salt is apharmaceutically acceptable salt, although this is not required forsalts of intermediate compounds that are not intended for administrationto a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), triisopropylsiliyl (TIPS),tert-butyldimethylsilyl (TBS or TBDMS),[2-(trimethylsilyl)-ethoxy]methyl (SEM); and the like. Numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York

General Synthetic Procedures

Compounds of this invention, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., R¹, R², R³, R⁴, etc.)used in the following schemes have the same meanings as those definedelsewhere herein unless otherwise indicated. Additionally, compoundshaving an acidic or basic atom or functional group may be used or may beproduced as a salt unless otherwise indicated (in some cases, the use ofa salt in a particular reaction will require conversion of the salt to anon-salt form, e.g., a free base, using routine procedures beforeconducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials known to those skilled in the art. Inparticular, it will be appreciated that compounds of the invention maybe prepared by a variety of process routes in which reactants arecombined in different orders to provide different intermediates en routeto producing final products.

A general method of preparing final compounds of the invention utilizesa key intermediate 1 as illustrated in Scheme 1. The variables R¹, R²,R⁴, R⁵, and n are defined as in formula (I), R^(A) represents anoptionally substituted C₁₋₄alkyl, and L is a leaving group. The schemeshows compounds in which the variable R³ is methyl. Compounds in whichR³ is C₂₋₃alkyl may be prepared analogously.

Sulfonamide compounds in which R¹ is defined as —S(O)₂R⁴ as in option(a) are typically prepared by contacting intermediate 1 with betweenabout 1 and about 1.1 equivalents of a sulfonylchloride of the formCl—S(O)₂R⁴ in the presence of an excess of base at a temperature on theorder of 0° C. The reaction is typically conducted for between about 1and about 24 hours or until the reaction is substantially complete.

To prepare compounds in which R¹ is an optionally substituted alkylgroup as defined in option (b), the alkylation reaction typically uses ahalo leaving group L, principally chloro or bromo. The reaction istypically conducted by contacting intermediate 1 with an excess of thereagent L-R^(A) in an inert diluent in the presence of an excess ofbase. The reaction is typically conducted at a temperature between about20° C. and about 60° C. for between about 10 and about 24 hours or untilthe reaction is substantially complete.

Alternatively, the Michael addition reaction may be used to preparecompounds in which R¹ is a cyanoethyl group. For example, as describedin the examples below, to prepare a compound in which R¹ is —(CH₂)₂CN,intermediate 1 is contacted with between about 1 and about 1.5equivalents of acrylonitrile in the presence of an excess of base, forexample diisopropylethylamine or diazobicycloundecene. The reaction istypically conducted at room temperature for between about 3 and about 24hours or until the reaction is substantially complete.

Compounds in which R¹ is defined as —C(O)R⁵ may be prepared using acarbonyl chloride of the form Cl—C(O)R⁵, specifically a chloroformatewhen R⁵ is defined as —OC₁₋₄alkyl. Typically, intermediate 1 iscontacted with about one equivalent of the carbonyl chloride in thepresence of an excess of base at a temperature on the order of 0° C. Thereaction is typically conducted for between about 1 and about 3 hours oruntil the reaction is substantially complete.

Alternatively, compounds in which R¹ is defined as —C(O)R⁵ may beprepared by contacting intermediate 1 with a modest excess of carboxylicacid reagent HO—C(O)—R⁵ under typical amide coupling conditions. Thereaction is typically performed in the presence of an excess of baseutilizing an activating agent such asN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU). The reaction is typically conducted at roomtemperature for between about 3 and about 24 hours or until the reactionis substantially complete.

An exemplary reaction for the preparation of intermediate 1 in which thevariable R³ is methyl is illustrated in Scheme 2.

In the aromatic substitution reaction of step 1, the trichloropyrimidine2 is reacted with an excess of the amino-pyrazole-methanol intermediate3 in the presence of base to provide intermediate 4. The Boc-protectedamino-aza-bicyclo intermediate 5 is then reacted with intermediate 4 toprovide intermediate 6. For example, intermediate 4 is combined withbetween about 1 and about 1.5 equivalents of the aza-bicyclointermediate 5 in the presence of an excess of base, such asdiisopropylethylyamine. The reaction is typically conducted at elevatedtemperature, between about 85° C. and about 120° C. for between about 6and about 12 hours or until the reaction is substantially complete.Reaction of intermediate 6 with sodium methoxide provides intermediate7. The reaction is typically conducted in a sealed tube at elevatedtemperature, between about 85° C. and about 120° C. for between about 4and about 10 hours or until the reaction is substantially complete. Inthe last step, the Boc group may be removed by standard treatment withan acid, typically hydrochloric acid, to provide intermediate 1.

Alternatively, intermediate 1 may be prepared by the sequence of stepsillustrated in Scheme 3.

where R^(B) is hydrogen or a silyl oxygen-protecting group, such astriisopropylsilyl (TIPS) or tert-butyldimethylsilyl (TBS). TheBoc-protected amino-aza-bicylo group 5 is combined with thedichloro-methoxypyrimidine intermediate 8 to form intermediate 9. Thereaction is typically conducted at elevated temperature in the presenceof base. Intermediate 9 is then reacted with amino-pyrazole intermediate3′ under standard Buchwald conditions to provide intermediate 7. Forexample, intermediate 9 is combined with between about 1 and about 1.5equivalents of the pyrazole intermediate 3′ in the presence of a basesuch as cesium carbonate or potassium carbonate and a palladiumcatalyst. The reaction is typically conducted at elevated temperature,between about 80° C. and about 110° C., for between about 8 and about 24hours or until the reaction is substantially complete. In the finalstep, the Boc protecting group is removed as in Scheme 2. When R^(B) isa silyl protecting group, the silyl and Boc groups can be removedsimultaneously.

Accordingly, in a method aspect, the invention provides a method forpreparing a compound of formula (I) or a pharmaceutically acceptablesalt thereof, the method comprising:

reacting a compound of formula (III):

with

-   -   (i) Cl—S(O)₂R⁴,    -   (ii) a compound of formula L-R^(A) wherein L is a leaving group        and R^(A) is C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally        substituted with —CN,

or pyridinyl, wherein pyridinyl is optionally substituted with —CN;

-   -   (iii) Cl—C(O)R⁵, or    -   (iv) HO—C(O)R⁵        wherein R¹, R², R³, R⁴, R⁵, and n are as defined above, and

optionally forming a pharmaceutically-acceptable salt

to provide a compound of formula (I) or a pharmaceutically acceptablesalt thereof.

In separate and distinct aspects, the invention provides a compound offormula (III) wherein the variables take any of the values describedabove and a compound of formula (III) wherein R² and R³ are each methyland n is 1 or 2.

In another method aspect, the invention provides a method for preparinga compound of formula 1

wherein R² and n are as defined above, the method comprising:

(a) reacting a compound of formula 9:

with a compound of formula 3:

wherein R^(B) is hydrogen or a silyl oxygen-protecting group to form acompound of formula 7:

and

(b) removing the protecting group or groups from the compound of formula7 to provide the compound of formula 1.

Crystalline Forms

In another aspect, the invention provides1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile(see Examples 2 and 10-13) in crystalline freebase Form I and Form II.

In one aspect, crystalline freebase Form I is characterized by a powderX-ray diffraction (PXRD) pattern having significant diffraction peaks,among other peaks, at 2θ values of 8.89±0.20, 12.99±0.20, 13.44±0.20,and 20.16±0.20. Form I may be further characterized by a PXRD patternhaving two or more additional diffraction peaks, including three or moreand four or more additional diffraction peaks at 20 values selected from10.64±0.20, 10.99±0.20, 15.02±0.20, 15.74±0.20, 16.47±0.20, 20.93±0.20,22.22±0.20, and 26.25±0.20. In another aspect, Form I is characterizedby a PXRD pattern having diffraction peaks at 20 values of 8.89±0.20,10.64±0.20, 10.99±0.20, 12.99±0.20, 13.44±0.20, 15.02±0.20, 15.74±0.20,16.47±0.20, 20.16±0.20, 20.93±0.20, 22.22±0.20, and 26.25±0.20.

As is well known in the field of powder X-ray diffraction, peakpositions of PXRD patterns are relatively less sensitive to experimentaldetails, such as details of sample preparation and instrument geometry,than are the relative peak heights. Thus, in one aspect, the crystallineForm I is characterized by a powder X-ray diffraction pattern in whichthe peak positions are substantially in accordance with those shown inFIG. 1.

In another aspect, crystalline Form I is characterized by its behaviorwhen exposed to high temperature. As demonstrated in FIG. 2, thedifferential scanning calorimetry (DSC) trace recorded at a heating rateof 10° C. per minute exhibits a peak in endothermic heat flow,identified as a melt transition, in the range of about 235° C. to about245° C., including between about 237° C. and about 242° C. The thermalgravimetric analysis (TGA) trace of FIG. 3 exhibits an onset of weightloss corresponding to post-melting decomposition.

Crystalline Form I has been demonstrated to have a reversiblesorption/desorption profile with an exceptionally small propensity forhygroscopicity. Form I demonstrated less than about 0.4% weight gain inthe humidity range of 5% to 90% relative humidity. No hysteresis wasobserved in two cycles of sorption and desorption. Form I is consideredto be non-hygroscopic.

In addition, crystalline Form I has been demonstrated to be stable tomicronization. No difference could be observed between the powder X-raydiffraction pattern of material that had not been micronized and thepattern of material of Form I after micronization.

Crystalline freebase Form II is characterized by the PXRD pattern ofFIG. 5 and further by its behavior when exposed to high temperature. Asdemonstrated in FIG. 6, the differential scanning calorimetry (DSC)trace recorded at a heating rate of 10° C. per minute exhibits a broadpeak in endothermic heat flow in the range of about 205° C. to about240° C., which, together with the thermal gravimetric analysis (TGA)trace of FIG. 7, may be interpreted as a merged melting transition anddecomposition. Form II is a slightly hygroscopic solid whichdemonstrated small hysteresis between two cycles of sorption anddesorption. Form II demonstrated about 1.2% weight gain in the humidityrange of 5% to 90% relative humidity.

As described in Examples 11 and 12, Form I may be prepared by dissolvingthe compound in N-methylpyrrolidone (NMP) or dimethylformamide (DMF) andadding acetone and water as antisolvents in a ratio of about 1:1.5 to1:1.75 acetone:water. The resulting reaction mixture is stirred forbetween about 4 hours and about 24 hours, filtered, washed with amixture of acetone and water, such as a 1:1.4 mixture of acetone andwater, and dried to provide crystalline Form I. A process for preparingcrystalline Form II is described in Example 13.

In another aspect, the invention provides a method of preparingcrystalline Form I, the method comprising: (a) dissolving1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrilein a diluent selected from N-methylpyrrolidone and dimethylformamide toform a reaction mixture; (b) adding acetone and water to the reactionmixture; and (c) isolating crystalline Form I from the reaction mixture.

Pharmaceutical Compositions

The compounds of the invention and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Such pharmaceutical compositions may be administered toa patient by any acceptable route of administration including, but notlimited to, oral, topical (including transdermal), rectal, nasal,inhaled, and parenteral modes of administration.

Accordingly, in one of its composition aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), where, as defined above, “compound of formula (I)” means acompound of formula (I) or a pharmaceutically-acceptable salt thereof.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired. When discussingcompositions and uses thereof, the “compound of the invention” may alsobe referred to herein as the “active agent”. As used herein, the term“compound of the invention” is intended to include all compoundsencompassed by formula (I) as well as the species embodied in formula(II) and pharmaceutically-acceptable salts thereof.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present invention.Those skilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the active agent; including from about 5 toabout 70% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise the active agent and one ormore pharmaceutically-acceptable carriers. Optionally, such solid dosageforms may comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as crosscarmellose sodium, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid, methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methylcellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The compounds of this invention can also be administered parenterally(e.g. by intravenous, subcutaneous, intramuscular or intraperitonealinjection). For parenteral administration, the active agent is typicallyadmixed with a suitable vehicle for parenteral administration including,by way of example, sterile aqueous solutions, saline, low molecularweight alcohols such as propylene glycol, polyethylene glycol, vegetableoils, gelatin, fatty acid esters such as ethyl oleate, and the like.Parenteral formulations may also contain one or more anti-oxidants,solubilizers, stabilizers, preservatives, wetting agents, emulsifiers,buffering agents, or dispersing agents. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention may also be formulated for topicaladministration to the skin as an ointment or cream. Ointmentformulations are semisolid preparations having a base of an oily orgreasy material that is typically clear. Suitable oily materials for usein ointment formulations include petrolatum (petroleum jelly), beeswax,cocoa butter, shea butter, and cetyl alcohol. Ointments may optionallyadditionally include emollients and penetration enhancers, if desired.

Cream formulations may be prepared as emulsions comprising an oil phaseand aqueous phase, typically including purified water. Components ofcream formulations may include: oil bases, such as petrolatrum, mineraloils, vegetable and animal oils, and triglycerides; cream bases, such aslanolin alcohols, stearic acid, and cetostearyl alcohol; a gel base,such as polyvinyl alcohol; solvents, such as, propylene glycol andpolyethylene glycol; emulsifiers, such as polysorbates, stearates, suchas glyceryl stearate, octylhydroxystearate, polyoxyl stearate, PEGstearyl ethers, isopropyl palmitate, and sorbitan monostearate;stabilizers, such as polysaccharides and sodium sulfite; emollients(i.e.moisturizers), such as medium chain triglycerides, isopropylmyristate, and dimethicone; stiffening agents, such as cetyl alcohol andstearyl alcohol; antimicrobial agents, such as methylparaben,propylparaben, phenoxyethanol, sorbic acid, diazolidinyl urea, andbutylated hydroxyanisole; penetration enhancers, such asN-methylpyrrolidone, propylene glycol, polyethylene glycol monolaurate,and the like; and chelating agents, such as edetate disodium.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is dry blended with microcrystalline cellulose, polyvinylpyrrolidone, and croscarmellose sodium in a ratio of 4:5:1:1 andcompressed into tablets to provide a unit dosage of, for example, 5 mg,20 mg or 40 mg active agent per tablet.

Capsule Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is combined with microcrystalline cellulose, polyvinylpyrrolidone, and crosscarmellose sodium in a ratio of 4:5:1:1 by wetgranulation and loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 5 mg, 20 mg or 40 mgactive agent per capsule.

Liquid Formulation

A liquid formulation comprising a compound of the invention (0.1%),water (98.9%) and ascorbic acid (1.0%) is formed by adding a compound ofthe invention to a mixture of water and ascorbic acid.

Enteric Coated Oral Dosage Form

A compound of the invention is dissolved in an aqueous solutioncontaining polyvinyl pyrrolidone and spray coated onto microcrystallinecellulose or sugar beads in a ratio of 1:5 w/w active agent:beads andthen an approximately 5% weight gain of an enteric coating comprising anacrylic copolymer, for example a combination of acrylic copolymersavailable under the trade names Eudragit-L® and Eudragit-S®, orhydroxypropyl methylcellulose acetate succinate is applied. The entericcoated beads are loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 30 mg active agentper capsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Ointment Formulation for Topical Administration

A compound of the invention is combined with petrolatum, C₈-C₁₀triglyceride, octylhydroxystearate, and N-methylpyrrolidone in a ratioto provide a composition containing 0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

A compound of the invention is combined with white petrolatum, propyleneglycol, mono- and di-glycerides, paraffin, butylated hydroxytoluene, andedetate calcium disodium in a ratio to provide a composition containing0.05% to 5% active agent by weight.

Ointment Formulation for Topical Administration

A compound of the invention is combined with mineral oil, paraffin,propylene carbonate, white petrolatum and white wax to provide acomposition containing 0.05% to 5% active agent by weight.

Cream Formulation for Topical Administration

Mineral oil is combined with a compound of the invention, propyleneglycol, isopropyl palmitate, polysorbate 60, cetyl alcohol, sorbitanmonostearate, polyoxyl 40 stearate, sorbic acid, methylparaben andpropylparaben to form an oil phase, which is combined with purifiedwater by shear blending to provide a composition containing 0.05% to 5%active agent by weight.

Cream Formulation for Topical Administration

A cream formulation comprising a compound of the invention, benzylalcohol, cetyl alcohol, citric acid anhydrous, mono and di-glycerides,oleyl alcohol, propylene glycol, sodium cetostearyl sulphate, sodiumhydroxide, stearyl alcohol, triglycerides, and water contains 0.05% to5% active agent by weight.

Cream Formulation for Topical Administration

A cream formulation comprising a compound of the invention, cetostearylalcohol, isopropyl myristate, propylene glycol, cetomacrogol 1000,dimethicone 360, citric acid, sodium citrate, and purified water, withimidurea, methylparaben, and propylparaben, as preservatives, contains0.05% to 5% active agent by weight.

Utility

The compounds of the invention have been shown to be potent inhibitorsof the JAK family of enzymes: JAK1, JAK2, JAK3, and TYK2. Inhibition ofthe family of JAK enzymes could inhibit signaling of many keypro-inflammatory cytokines. Thus the JAK inhibitors of the invention areexpected to be useful in the treatment of inflammatory diseases such asulcerative colitis, and other gastrointestinal inflammatory diseasessuch as Crohn's disease and immune checkpoint inhibitor induced colitis.The present JAK inhibitors are also expected to be useful in thetreatment of atopic dermatitis and other inflammatory and pruritic skindiseases and in the treatment of respiratory conditions such as allergicrhinitis, asthma, and chronic obstructive pulmonary disease (COPD).

Gastrointestinal Inflammatory Disease

In addition to providing potent inhibition of JAK enzymes, compounds ofthe invention have been designed to be poorly absorbed to minimizesystemic exposure. Selected compounds tested in cannulated rats showedlow oral bioavailability. In addition, the compounds are designed tohave their effect at the site of action, for example, in the colon. Asdescribed in Assays 6 and 7 below, the compound of Example 2 exhibitedoral bioavailability in rat less than about 5% and a ratio of exposurein the colon to exposure in plasma upon oral administration greater thanabout 250.

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis. As describedbelow, the compound of Example 2, among other compounds of theinvention, demonstrated activity in the oxazolone-induced colitis modelin mice. Further, when tested in an immunosuppression model in mice,which probes systemic functional activity, the compound demonstratedminimal effect of immunosuppression at the same dose required todemonstrate efficacy in the oxazolone model. Thus the compounddemonstrated anti-colitic activity without exhibiting systemic effectsin preclinical models.

It is expected that a high colon to plasma ratio will provide robust,luminally-driven anti-inflammatory activity without associated,systemically-driven, adverse effects. Such compounds are expected to beuseful for a variety of gastrointestinal inflammatory indications thatinclude, but are not limited to, ulcerative colitis (proctosigmoiditis,pancolitis, ulcerative proctitis and left-sided colitis), Crohn'sdisease, collagenous colitis, lymphocytic colitis, Behcet's disease,celiac disease, immune checkpoint inhibitor induced colitis, ileitis,eosinophilic esophagitis, graft versus host disease-related colitis, andinfectious colitis. Ulcerative colitis (Reimund et al., J ClinImmunology, 1996, 16, 144-150), Crohn's disease (Woywodt et al., Eur JGastroenterology Hepatology, 1999, 11, 267-276), collagenous colitis(Kumawat et al., Mol Immunology, 2013, 55, 355-364), lymphocytic colitis(Kumawat et al., 2013), eosinophilic esophagitis (Weinbrand-Goichberg etal., Immunol Res, 2013, 56, 249-260), graft versus host disease-relatedcolitis (Coghill et al., Blood, 2001, 117, 3268-3276), infectiouscolitis (Stallmach et al., Int J Colorectal Dis, 2004, 19, 308-315),Behcet's disease (Zhou et al., Autoimmun Rev, 2012, 11, 699-704), celiacdisease (de Nitto et al., World J Gastroenterol, 2009, 15, 4609-4614),immune checkpoint inhibitor induced colitis (e.g., CTLA-4inhibitor-induced colitis; (Yano et al., J Translation Med, 2014, 12,191), PD-1- or PD-L1-inhibitor-induced colitis), and ileitis (Yamamotoet al., Dig Liver Dis, 2008, 40, 253-259) are characterized by elevationof certain pro-inflammatory cytokine levels. As many pro-inflammatorycytokines signal via JAK activation, compounds described in thisapplication may be able to alleviate the inflammation and providesymptom relief.

In particular, the compounds of the invention are expected to be usefulfor the induction and maintenance of remission of ulcerative colitis,and for the treatment of Crohn's disease, immune checkpoint inhibitorinduced colitis, and the gastrointestinal adverse effects in graftversus host disease.

In one aspect, therefore, the invention provides a method of treating agastrointestinal inflammatory disease in a mammal (e.g., a human), themethod comprising administering to the mammal a compound of theinvention or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

The invention further provides a method of treating ulcerative colitisin a mammal, the method comprising administering to the mammal acompound of the invention or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

When used to treat ulcerative colitis, the compounds of the inventionwill typically be administered orally in a single daily dose or inmultiple doses per day, although other forms of administration may beused. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating ulcerative colitis and othergastrointestinal inflammatory disorders are expected to range from about1 to about 400 mg/day of active agent, including from about 5 to about300 mg/day and from about 20 to about 70 mg per day of active agent foran average 70 kg human.

Combination Therapy

Compounds of the invention may also be used in combination with one ormore agents which act by the same mechanism or by different mechanismsto effect treatment of gastrointestinal inflammatory disorders. Usefulclasses of agents for combination therapy include, but are not limitedto, aminosalicylates, steroids, systemic immunosuppressants, anti-TNFαantibodies, anti-VLA-4 antibodies, anti-integrin α₄β₇ antibodies,anti-bacterial agents, and anti-diarrheal medicines.

Aminosalicylates that may be used in combination with the present JAKinhibitor compounds include, but are not limited to, mesalamine,osalazine and sulfasalazine. Examples of steroids include, but are notlimited to, prednisone, prednisolone, hydrocortisone, budesonide,beclomethasone, and fluticasone. Systemic immunosuppressants useful fortreatment of inflammatory disorders include, but are not limited tocyclosporine, azathioprine, methotrexate, 6-mercaptopurine, andtacrolimus. Further, anti-TNFα antibodies, which include, but are notlimited to, infliximab, adalimumab, golimumab, and certolizumab, may beused in combination therapy. Useful compounds acting by other mechanismsinclude anti-VLA-4 antibodies, such as natalizumab, anti-integrin α₄β₇antibodies, such as vedolizumab, anti-bacterial agents, such asrifaximin, and anti-diarrheal medicines, such as loperamide. (Mozaffariet al. Expert Opin. Biol. Ther. 2014, 14, 583-600; Danese, Gut, 2012,61, 918-932; Lam et al., Immunotherapy, 2014, 6, 963-971.)

In another aspect, therefore, the invention provides a therapeuticcombination for use in the treatment of gastrointestinal inflammatorydisorders, the combination comprising a compound of the invention andone or more other therapeutic agents useful for treatinggastrointestinal inflammatory disorders. For example, the inventionprovides a combination comprising a compound of the invention and one ormore agents selected from aminosalicylates, steroids, systemicimmunosuppressants, anti-TNFα antibodies, anti-VLA-4 antibodies,anti-integrin α₄β₇ antibodies, anti-bacterial agents, and anti-diarrhealmedicines. Secondary agent(s), when included, are present in atherapeutically effective amount, i.e. in any amount that produces atherapeutically beneficial effect when co-administered with a compoundof the invention.

Also provided, therefore, is a pharmaceutical composition comprising acompound of the invention and one or more other therapeutic agentsuseful for treating gastrointestinal inflammatory disorders.

Further, in a method aspect, the invention provides a method of treatinggastrointestinal inflammatory disorders, the method comprisingadministering to the mammal a compound of the invention and one or moreother therapeutic agents useful for treating gastrointestinalinflammatory disorders.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, as disclosed above, or the agents maybe provided in separate compositions that are administeredsimultaneously or at separate times, by the same or by different routesof administration. When administered separately, the agents areadministered sufficiently close in time so as to provide a desiredtherapeutic effect. Such compositions can be packaged separately or maybe packaged together as a kit. The two or more therapeutic agents in thekit may be administered by the same route of administration or bydifferent routes of administration.

Inflammatory Skin Disease

Atopic dermatitis, for example, has been associated with elevation ofproinflammatory cytokines that rely on the JAK-STAT pathway, inparticular, IL-4, IL-5, IL-10, IL-13, and IFNγ. Since compounds of theinvention exhibit potent inhibition at all four JAK enzymes, they areexpected to potently inhibit the proinflammatory cytokinescharacteristic of atopic dermatitis and other inflammatory skindiseases. In particular, the compound1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,disclosed in Example 2, exhibited IC₅₀ values of 50 nM or less forinhibition of IL-4, IL-13, and IFNγ in the cellular assays described inAssays 4, 2, and 5, respectively. The compound also exhibited a dose-and concentration-dependent effect in a TPA-induced irritant contactdermatitis model in mice. Furthermore, model cream and ointmentformulations of the compound of Example 2 have demonstrated sustaineddermal levels for at least 2 days in mice and at least 7 days inmini-pig without detectable plasma exposure.

It is expected that sustained dermal levels of JAK inhibitors in theabsence of significant systemic levels will result in potent localanti-inflammatory and anti-pruritic activity in the skin withoutsystemically-driven adverse effects. Such compounds are expected to bebeneficial in a number of dermal inflammatory or pruritic conditionsthat include, but are not limited to atopic dermatitis, alopecia areata,vitiligo, cutaneous T cell lymphoma, prurigo nodularis, lichen planus,primary localized cutaneous amyloidosis, bullous pemphigoid, skinmanifestations of graft versus host disease, pemphigoid, discoid lupus,granuloma annulare, lichen simplex chronicus, vulvar/scrotal/perianalpruritus, lichen sclerosus, post herpetic neuralgia itch, lichenplanopilaris, and foliculitis decalvans. In particular, atopicdermatitis (Bao et al., JAK-STAT, 2013, 2, e24137), alopecia areata(Xing et al., Nat Med. 2014, 20, 1043-1049), vitiligo (Craiglow et al,JAMA Dermatol. 2015, 151, 1110-1112), cutaneous T cell lymphoma(Netchiporouk et al., Cell Cycle. 2014; 13, 3331-3335), prurigonodularis (Sonkoly et al., J Allergy Clin Immunol. 2006, 117, 411-417),lichen planus (Welz-Kubiak et al., J Immunol Res. 2015, ID:854747),primary localized cutaneous amyloidosis (Tanaka et al., Br J Dermatol.2009, 161, 1217-1224), bullous pemphigoid (Feliciani et al., Int JImmunopathol Pharmacol. 1999, 12, 55-61), and dermal manifestations ofgraft versus host disease (Okiyama et al., J Invest Dermatol. 2014, 134,992-1000) are characterized by elevation of certain cytokines thatsignal via JAK activation. Accordingly, compounds of the invention maybe able to alleviate associated dermal inflammation or pruritus drivenby these cytokines. In particular, compounds of the invention areexpected to be useful for the treatment of atopic dermatitis and otherinflammatory skin diseases.

In one aspect, therefore, the invention provides a method of treating aninflammatory skin disease in a mammal (e.g., a human), the methodcomprising applying a pharmaceutical composition comprising a compoundof the invention and a pharmaceutical carrier to the skin of the mammal.In one aspect, the inflammatory skin disease is atopic dermatitis.

Compounds of the invention may also be used in combination with grampositive antibiotics, such as mupirocin and fusidic acid, to treatinflammatory skin disease. In one aspect, therefore, the inventionprovides a method of treating an inflammatory skin disease in a mammal,the method comprising applying a compound of the invention and a grampositive antibiotic to the skin of the mammal. In another aspect, theinvention provides a pharmaceutical composition comprising a compound ofthe invention, a gram positive antibiotic, and apharmaceutically-acceptable carrier.

Compounds of the invention have been demonstrated to be potentinhibitors of the JAK1, JAK2, JAK3, and TYK2 enzymes in enzyme bindingassays and to have potent functional activity without cytotoxicity incellular assays, as described in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   ACN=acetonitrile    -   CPME=cyclopentyl methyl ether    -   d=day(s)    -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   EtOH=ethyl alcohol    -   h=hour(s)    -   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   IPA=isopropyl alcohol    -   MeOH=methanol    -   min=minute(s)    -   NMP=N-methylpyrrolidone    -   RT=room temperature    -   TEA=triethylamine    -   THF=tetrahydrofuran    -   TFA=trifluoroacetic acid    -   Xantphos=4,5-bis(diphenylphosphino)-9,9-dimethylxanthene    -   Xphos=dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl    -   XphosPd        G2=chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyO]palladium(II)

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Unless otherwise indicated the following conditions were used forpreparative HPLC purifications.

-   Column: C18, 5 μm 21.2×150 mm or C18, 5 μm 21×250 mm or C14, 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,-   Injection volume: (100-15004)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytic HPLC Conditions Method A

-   Column: LUNA C18 (2), 150×4.60 mm, 3 μm-   Column temperature: 37° C.-   Flow rate: 1.0 mL/min-   Injection volume: 5 μL-   Sample preparation: Dissolve in 1:1 ACN:water-   Mobile Phases: A=Water:ACN:TFA (98:2:0.05)    -   B=Water:ACN:TFA (2:98:0.05)-   Detector wavelength: 250 nm-   Gradient: 32 min total (time (min)/% B): 0/2, 10/20, 24/90, 29/90,    30/2, 32/2

Method B

-   Column: LUNA C18 (2), 150×4.60 mm, 3 μm-   Column temperature: 37° C.-   Flow rate: 1.0 mL/min-   Injection volume: 10 μL-   Sample preparation: Dissolve in 1:1 ACN:water-   Mobile Phases: A=Water:ACN:TFA (98:2:0.05)    -   B=Water:ACN:TFA (10:90:0.05)-   Detector wavelength: 254 nm-   Gradient: 35 min total (time (min)/% B): 0/2, 20/25, 23/90, 26/90,    27/2, 35/2

Preparation 1:(3-((2,6-Dichloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

To a mixture of 2,4,6-trichloropyrimidine (8.0 g, 43.7 mmol) and(3-amino-1H-pyrazol-5-yl)methanol (7.4 g, 65.4 mmol) in EtOH (80 mL) wasadded DIPEA (11.3 g, 87.2 mmol). The reaction mixture was stirred at 20°C. for 12 h and filtered to give the title intermediate (6.5 g, 57%yield) as a white solid. (m/z): [M+H]⁺ calcd for C₈H₇Cl₂N₅O 260.00 found260.0.

Preparation 2:(3-((2-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

(a) tert-Butyl(1R,3s,5S)-3-((4-chloro-6-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A mixture of(3-((2,6-dichloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol (6.0 g,23.1 mmol), tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (6.3 g, 27.7mmol), and DIPEA (6.0 g, 46.2 mmol) in DMSO (60 mL) was stirred at 100°C. for 12 h. The reaction mixture was combined with the product of apilot scale run and poured into water (800 mL). The precipitate wasfiltered and dried in vacuo. The residue was re-crystallized from EtOAc(500 mL) and petroleum ether (500 mL) to afford the title intermediate(5.6 g, 50% yield) as a gray solid. Structure confirmed by NMR.

(b) tert-Butyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a solution of sodium methoxide (2.4 g, 44.5 mmol) in MeOH (30 mL) wasadded the product of the previous step (2.0 g, 4.45 mmol) at 20° C. Thereaction mixture was stirred in a sealed tube at 100° C. for 8 h. Thereaction mixture was combined with the product of a pilot scale run,poured into water (30 mL), and extracted with EtOAc (3×50 mL). Theorganic layer was washed with brine (2×30 mL), dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified bypreparative HPLC (Daiso 250×50 mm 10 μm, 80 mL/min, 30-55% ACN+0.1%TFA/ACN) to afford the title intermediate (0.7 g, 32% yield) as a whitesolid. (m/z): [M+H]⁺ calcd for C₂₁H₃₁N₇O₄446.24 found 446.2.

(c)(3-((2-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

A solution of the product of the previous step (0.7 g, 1.57 mmol) in 4 MHCl in EtOAc (20 mL) was stirred at 20° C. for 2 h, and concentrated invacuo to provide the HCl salt of the title compound (0.6 g, 99% yield)as a white solid. (m/z): [M+H]⁺ calcd for C₁₆H₂₃N₇O₂ 346.19 found 346.2.

Preparation 3: tert-Butyl(1R,3s,5S)-3-(methylamino)-8-azabicyclo[3.2.1]octane-8-carboxylate

(a) tert-butyl(1R,3s,5S)-3-(((benzyloxy)carbonyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A solution of tert-butyl(1R,3s,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (5.21 g,23.00 mmol), DMF (115 ml), and triethylamine (6.41 mL, 46.0 mmol) wasstirred at RT for 15 min. Benzyl chloroformate (3.56 mL, 25.3 mmol) wasadded dropwise and the reaction mixture was stirred at RT for 3 h,quenched with water, and extracted with EtOAc (4×20 mL). The combinedorganic fractions were washed with brine (2×20 mL), dried over sodiumsulfate, filtered, and concentrated to afford a yellow oil which waspurified by column chromatography (120 g column; 0-70% EtOAc in hexanes)to afford the title intermediate as a thick, clear oil (3.79 g, 36%yield; 79% purity). (m/z): [M+H]⁺ calcd for C₂₀H₂₈N₂O₄ 361.20 found361.2.

(b)tert-butyl(1R,3s,5S)-3-(((benzyloxy)carbonyl)(methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A solution of the product of the previous step (2.99 g, 8.29 mmol) inDMF (41.5 mL) was cooled to 0° C. and sodium hydride, 60% dispersion inmineral oil (0.398 g, 16.58 mmol) was added in one portion. Theresulting suspension was stirred at 0° C. for 15 min, and theniodomethane (1.03 mL, 16.58 mmol) was added dropwise and the resultingcloudy, pale yellow mixture was stirred at 0° C. for 15 min, warmed toRT and stirred for 2 h. The reaction mixture was quenched with water andextracted with EtOAc (4×20 mL). The combined organic fractions werewashed with brine (2×20 mL), dried over sodium sulfate, filtered, andconcentrated to afford a clear, pale yellow oil, which was purified bycolumn chromatography (80 g column; 0-70% EtOAc in hexanes) to affordthe title intermediate as a clear, colorless thick oil (2.07 g, 65%yield; 97% purity). (m/z): [M+H]⁺ calcd for C₂₁H₃₀N₂O₄ 375.22 found375.5.

(c) tert-Butyl(1R,3s,5S)-3-(methylamino)-8-azabicyclo[3.2.1]octane-8-carboxylate

To a 100 mL flask was added palladium, 10% wt. on carbon (0.577 g, 0.542mmol). The material was exposed to nitrogen and then a solution of theproduct of the previous step (1.015 g, 2.71 mmol) in MeOH (54.2 mL) wasslowly added by pipette. A hydrogen gas balloon was attached. The flaskwas evacuated and back-filled with hydrogen three times before openingthe atmosphere fully to H₂ gas. The reaction mixture was stirred at RTfor 16 h, filtered through a pad of Celite®, and concentrated to afforda clear oil, which was purified by column chromatography 40 g column;0-100% MeOH in DCM) to afford the product as a clear oil. The column wasflushed with 10:1 MeOH:TEA. The filtrate was concentrated to provide athick, clear oil with white solid which was dissolved in EtOAc,filtered, combined with the clear oil product and concentrated toprovide the title intermediate (0.559 g, 86% yield) (m/z): [M+H]⁺ calcdfor C₁₃H₂₄N₂O₂ 241.18 found 241.3.

Preparation 4:(3-((2-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

(a) tert-Butyl(1R,3s,5S)-3-((4-chloro-6-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate

A solution of(3-((2,6-dichloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol (200 mg,0.77 mmol), tert-butyl(1R,3s,5S)-3-(methylamino)-8-azabicyclo[3.2.1]octane-8-carboxylate (194mg, 0.81 mmol) and TEA (0.29 mL, 1.92 mmol) were stirred in DMSO (5 mL)overnight at 60° C. The reaction mixture was concentrated in vacuo. Thecrude residue was purified by reverse phase chromatography to afford thetitle intermediate (143 mg, 0.31 mmol, 40% yield), which was useddirectly in the next step.

(b)(3-((2-(((1R,3s,5S)-8-Azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-6-chloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

To the product of the previous step (143 mg, 0.31 mmol) dissolved in ACN(3.0 mL) was added 4 N HCl in dioxane (1.156 mL 4.62 mmol) and thereaction mixture was stirred at RT for 30 min. The reaction mixture wasconcentrated in vacuo to provide the HCl salt of the title intermediatewhich was used without purification in the next step.

(c)(3-((2-(((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

To a stirred solution of the product of the previous step (112 mg, 0.280mmol) in MeOH (5 mL) was added 50% sodium methoxide in MeOH (0.960 mL,8.39 mmol). The reaction mixture was heated in a sealed vial at 80° C.overnight. The reaction mixture was concentrated in vacuo and the cruderesidue was purified via reverse phase chromatography to afford thetitle product (27 mg, 0.057 mmol, 20% yield).

Preparation 5:(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

(a) tert-Butyl(1R,3s,5S)-3-((4-chloro-6-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a mixture of(3-((2,6-dichloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol (3.7 g,14.2 mmol) and tert-butyl(1R,3s,5S)-3-amino-9-azabicyclo[3.3.1]nonane-9-carboxylate (4.1 g, 17.0mmol) in DMSO (37 mL) was added DIPEA (3.7 g, 28.4 mmol) under nitrogen.The reaction was stirred at 120° C. for 12 h, poured into water (80 mL),extracted with EtOAc (3×100 mL), dried, and concentrated to providecrude product, which was washed with EtOAc (20 mL), to give the titleintermediate (3.8 g, 56% yield) as a white solid.

(b) tert-Butyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

Four reactions were carried out in parallel. To a mixture of the productof the previous step (0.95 g, 2.0 mmol) in sodium methoxide in MeOH (10mL) was stirred at 120° C. for 3 h in a sealed tube. The reactionmixture was added to water (50 mL) and extracted with EtOAc (3×50 mL).The organic layers were washed with brine (30 mL), dried over Na₂SO₄,and concentrated in vacuo. The residue was purified by preparative HPLC(Luna C18 250×50 mm 10 μm, ACN+0.1% TFA/ACN) to obtain the titleintermediate (1.2 g combined product, 28% yield) as a brown solid.(m/z): [M+H]⁺ calcd for C₂₂H₃₃N₇O₄ 460.26 found 460.3.

(c)(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

To the product of the previous step (1.2 g, 2.5 mmol) was added 4 M HClin EtOAc (50 mL). The reaction was stirred at 25° C. for 2 h. Theresidue was combined with the product of a preparation at the 1.5 mmolscale and concentrated to provide the title intermediate (2.0 g, 100%yield) as a light yellow solid. (m/z): [M+H]⁺ calcd for C₁₇H₂₅N₇O₂360.43 found 360.4.

Preparation 6:(3-((2-(((1R,3s,5S)-9-Azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

(a) tert-Butyl(1R,3s,5S)-3-((4-chloro-6-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a mixture of(3-((2,6-dichloropyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol (6.5 g,24.9 mmol), tert-butyl(1R,3s,5S)-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (6.9g, 27.4 mmol) in DMSO (80 mL) was added DIPEA (6.4 g, 49.8 mmol) undernitrogen. The reaction mixture was stirred at 120° C. for 8 h, pouredinto water (80 mL), extracted with EtOAc (500 mL), dried, andconcentrated to provide the crude product. The crude product wascombined with the product of a separate preparation at the same scaleand purified by preparative HPLC (Daiso 150×25 mm 5 μm, 80 mL/min,35-60% ACN+0.1% TFA/ACN) to provide the title intermediate (16.0 g, 64%yield) as a light yellow solid. (m/z): [M+H]⁺ calcd for C₂₂H₃₂C₁N₇O₃478.23 found 478.2.

(b) tert-Butyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a mixture of the product of the previous step (2.0 g, 4.19 mmol) inMeOH (20 mL) was added sodium methoxide (2.2 g, 41.9 mmol). The reactionmixture was stirred at 120° C. for 6 h in a sealed tube for 12 h, pouredinto water (100 mL), diluted with EtOAc (800 mL), washed with brine (50mL), dried, and concentrated to provide crude product. The crude productwas combined with the product of a separate preparation at the 2 mmolscale and purified by preparative HPLC (Synergi Max-RP, 250×50 mm 10 μm,80 mL/min, 25-50% ACN+0.1% TFA/ACN) to provide the title intermediate(2.1 g, 71% yield) as a white solid.

(c)(3-((2-(((1R,3s,5S)-9-Azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

To a mixture of the product of the previous step (2.1 g, 4.43 mmol) inEtOAc (20 mL) was added 4 M HCl in EtOAc (20 mL), and the reaction wasstirred at 20° C. for 3 h and concentrated to provide the HCl salt ofthe product (2.0 g, 100% yield) as a white solid. (m/z): [M+H]⁺ calcdfor C₁₈H₂₇N₇O₂ 374.22 found 374.1.

Example 1:1-(((1R,3s,5S)-3-((4-((5-(Hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)sulfonyl)azetidine-3-carbonitrile

To a solution of(3-((2-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol(15 mg, 0.042 mmol) in DMF (4.0 mL) was added DIPEA (0.022 ml, 0.125mmol) followed by 3-cyano-1-azetidinesulfonylchloride (7.54 mg, 0.042mmol). The reaction mixture was stirred at RT overnight. The solvent wasremoved in vacuo and the crude residue was purified by reverse-phaseHPLC to provide the TFA salt of the title compound (3.2 mg). (m/z):[M+H]⁺ calcd for C₂₁H₂₉N₉O₄S 504.21 found 504.1.

Example 2:1-(((1R,3s,5S)-3-((4-((5-(Hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile

To a solution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanolHCl (150 mg, 0.402 mmol) and DIPEA (0.351 mL, 2.008 mmol) in DMF (3 mL)at 0° C. was added 3-cyano-1-azetidinesulfonylchloride (72.5 mg, 0.402mmol) and the reaction mixture was stirred at 0° C. for 10 min and thenat RT for 15 h. The reaction mixture was concentrated in vacuo to yielda red liquid which was purified by preparative HPLC to yield the TFAsalt of the title compound (72.4 mg, 0.115 mmol, 28.5% yield) as a whitesolid. (m/z): [M+H]⁺ calcd for C₂₂H₃₁N₉O₄S 518.22 found 518.

Example 3:(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(methylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

A solution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanolHCl (250 mg, 0.669 mmol) in DMF (7.0 mL) was cooled to 0° C. and DIPEA(0.35 mL, 2.008 mmol) was added in one portion followed bymethanesulfonyl chloride (0.053 mL, 77 mg, 0.676 mmol) added dropwise.The reaction mixture was stirred overnight, dissolved in 1:1 aceticacid:water (6 mL), filtered, and purified by preparative HPLC to providethe TFA salt of the title compound (94 mg, 31% yield) as a white powder.(m/z): [M+H]⁺ calcd for C₁₉H₂₉N₇O₄S 452.15 found 452.2.

Example 4:(3-((6-methoxy-2-(((1R,3s,5S)-9-((2-methoxyethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol

A solution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanolHCl (250 mg, 0.703 mmol) in DMF (7.0 mL) was cooled to 0° C. and DIPEA(0.35 mL, 2.008 mmol) was added in one portion followed by2-methoxy-ethanesulfonyl chloride (111 mg, 0.676 mmol) added dropwise.The reaction mixture was stirred overnight, dissolved in 1:1 aceticacid:water (6 mL), filtered, and purified by preparative HPLC to providethe TFA salt of the title compound (41 mg, 12% yield) as a white solid.(m/z): [M+H]⁺ calcd for C₂₁H₃₃N₇O₅S 496.23 found 496.2.

Example 5:3-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)-propanenitrile

To a solution of(3-((2-((1R,3s,5S)-8-azabicyclo[3.2.1]octan-3-yl(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol(15 mg, 0.042 mmol) in MeOH (4.0 mL) was added DIPEA (0.022 mL, 0.125mmol) followed by acrylonitrile (2.70 μL, 0.042 mmol). The reactionmixture was stirred at RT overnight, concentrated in vacuo and purifiedby reverse-phase HPLC to provide the TFA salt of the title compound (4.3mg). (m/z): [M+H]⁺ calcd for C₂₀H₂₈N₈O₂ 413.23 found 413.2.

Example 6:5-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile

Diisopropylamine (0.032 mL, 0.225 mmol) (0.256 mL) was added to a 0.15 Msolution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol(29.8 mg, 0.075 mmol) in DMF and the solution was swirled to dissolveall the material. To the solution was added a 0.23 M solution of4-(chloromethyl)-picolinonitrile) (0.5 mL, 34 mg, 0.113 mmol) in DMF andthe reaction mixture was stirred at RT overnight. Polystyrene-thiophenolresin (0.115 g, 0.150 mmol) was added, the reaction mixture was stirredat RT for 4 h and filtered. The reaction vessel was washed with DMF (0.5mL) and the washes were combined, concentrated by rotary evaporation,dissolved in 1:1 acetic acid:water, filtered, and purified byreverse-phase HPLC to provide the TFA salt of the title compound (6.4mg). (m/z): [M+H]⁺ calcd for C₂₄H₂₉N₉O₂ 476.24 found 476.1.

Example 7:5-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)nicotinonitrile

A solution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanolHCl (20 mg, 0.054 mmol), 5-(bromomethyl)nicotinonitrile (10.55 mg, 0.054mmol) and potassium carbonate (22.20 mg, 0.161 mmol) were stirred in DMF(6.0 mL) at 60° C. overnight. The reaction mixture was concentrated invacuo and purified by reverse-phase HPLC to provide the TFA salt of thetitle intermediate (3.7 mg). (m/z): [M+H]⁺ calcd for C₂₅H₃₁N₉O₂ 490.26found 490.2.

Example 8: Isobutyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate

To a solution of(3-((2-((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,HCl (25 mg, 0.061 mmol) and DIPEA (42.6 μL, 0.244 mmol) in DMF (305 μL)at 0° C. was added isobutyl chloroformate (10 mg, 0.073 mmol) in DMF(305 μL) dropwise. The reaction mixture was stirred at 0° C. for 5 minand then allowed to reach RT. After 24 h, the reaction mixture wasconcentrated, dissolved in 1:1 ACN:water and purified by reverse-phaseHPLC to provide the TFA salt of the title compound (6.4 mg). (m/z):[M+H]⁺ calcd for C₂₃H₃₅N₇O₄ 474.28 found 474.2.

Example 9:2,2-difluoro-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one

HATU (0.029 g, 0.077 mmol) was added to a solution of 2,2-difluoroaceticacid (6.72 mg, 0.070 mmol) in DMF (3 mL) and the reaction mixture wasstirred at RT for 5 min to prepare a 0.14 M solution of the activatedacid. The 0.14 M solution of the activated acid (0.5 mL, 0.070 mmol) wasadded to a solution of(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanolHCl (29 mg, 0.070 mmol) and DIPEA (0.049 mL, 0.280 mmol) in DMF and thereaction mixture was stirred at RT for 30 min, concentrated in vacuo andthe resulting residue was dissolved in 1:1 acetic acid:water andpurified by reverse-phase HPLC to provide the TFA salt of the titlecompound (3.9 mg). (m/z): [M+H]⁺ calcd for C₂₀H₂₇N₇O₃ 452.21 found452.1.

Using similar synthetic methods, the compounds of Tables 1-3 wereprepared.

TABLE 1

Calc Found Ex No.  n  R² R⁴ Formula [M + H]⁺ [M + H]⁺ 1-1  1 Hazetidin-1-yl C₁₉H₂₈N₈O₄S 465.20 465   1-2  1 H

C₂₀H₂₇N₉O₄S 490.19 490   1-3  1 H pyridin-3-yl C₂₁H₂₆N₈O₄S 487.18 487.21-4  1 H phenyl C₂₂H₂₇N₇O₄S 486.18 486.3 1-5  2 H azetidin-1-ylC₂₀H₃₀N₈O₄S 479.21 479.1 1-6  2 H

C₂₁H₂₉N₉O₄S 504.21 504.1 1-7  2 H 5-fluoropyridin-3-yl C₂₂H₂₇FN₈O₄S519.19 519.1 1-8  2 H phenyl C₂₃H₂₉N₇O₄S 500.20 500.1 1-9  2 H —C₂H₅C₁₉H₂₉N₇O₄S 452.20 452.1 1-10 2 H —CH₂-cyclopropyl C₂₁H₃₁N₇O₄S 478.22478.1 1-11 2 H pyridin-3-yl C₂₂H₂₈N₈O₄S 501.20 501.1 1-12 2 —CH₃—(CH₂)₂CN C₂₁H₃₀N₈O₄S 491.21 491   1-13 2 —CH₃ pyrrolidin-1-ylC₂₂H₃₄N₈O₄S 507.24 507.2 1-14 2 —CH₃ cyclopropyl C₂₁H₃₁N₇O₄S 478.22478.1 1-15 2 —CH₃ pyridin-3-yl C₂₃H₃₀N₈O₄S 515.21 515.1 1-16 2 —CH₃phenyl C₂₄H₃₁N₇O₄S 514.22 514.1 1-17 2 —CH₃ azetidin-1-yl C₂₁H₃₂N₈O₄S493.23 493.2 1-18 2 —CH₃ —CH₂-cyclopropyl C₂₂H₃₃N₇O₄S 492.23 492.2 1-192 —CH₃ 5-fluoropyridin-3-yl C₂₃H₂₉FN₈O₄S 533.20 533.1

TABLE 2

Calc Found Ex No.  n  R² R¹ Formula [M + H]⁺ [M + H]⁺ 2-1 1 H—CH₂-pyridin-4-yl C₂₂H₂₈N₈O₂ 437.23 437   2-2 2 H —CH₂-pyridin-4-ylC₂₃H₃₀N₈O₂ 451.25 451.2 2-3 2 H

C₂₄H₂₉N₉O₂ 476.24 476.2 2-4 2 H —CH₂-pyridin-3-yl C₂₃H₃₀N₈O₂ 451.25451.2 2-5 2 —CH₃ —(CH₂)₂CN C₂₁H₃₀N₈O₂ 427.25 427   2-6 2 —CH₃

C₂₃H₃₂N₈O₂ 453.27 453   2-7 2 —CH₃ —CH₂-pyridin-4-yl C₂₄H₃₂N₈O₂ 465.27465.1 2-8 2 —CH₃

C₂₅H₃₁N₉O₂ 490.26 490.2

TABLE 3

Ex Calc Found No. n R² R⁵ Formula [M + H]⁺ [M + H]⁺ 3-1 2 H —CHF₂C₁₉H₂₅F₂N₇O₃ 438.20 438.1 3-2 2 —CH₃ —O-isobutyl C₂₃H₃₅N₇O₄ 474.28 474.23-3 2 —CH₃ —OCH₃ C₂₀H₂₉N₇O₄ 432.23 432.2 3-4 2 —CH₃ morpholin-C₂₃H₃₄N₈O₄ 487.27 487.2 1-yl 3-5 2 —CH₃ —CH₂- C₂₃H₃₃N₇O₃ 456.26 456.2cyclopropyl 3-6 2 —CH₃ —CH₃ C₂₀H₂₉N₇O₃ 416.23 416.1 3-7 2 —CH₃cyclopentyl C₂₄H₃₅N₇O₃ 470.28 470.2 3-8 2 —CH₃ cyclobutyl C₂₃H₃₃N₇O₃456.26 456.2

Example 10:1-(((1R,3s,5S)-3-((4-((5-(Hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile

(a) 5-(((triisopropylsilyl)oxy)methyl)-1H-pyrazol-3-amine (3′)

To a 100 mL flask was added (3-amino-1H-pyrazol-5-yl)methanol (5.8 g,51.3 mmol), 1-methyl-2-pyrrolidinone (58.0 mL) and imidazole (4.54 g,66.7 mmol) followed by triisopropylsilyl chloride (11.95 mL, 56.4mmol).The reaction mixture was stirred at 22° C. overnight and thenEtOAc (145 mL) and water (145 mL) were added. The layers were separatedand the organic layer was washed with water (145 mL) and brine (15%, 100mL), dried over Na₂SO₄, and evaporated under reduced pressure to providethe title intermediate (13.33 g, 49.5 mmol, 96% yield) HPLC Method ARetention time 19.00 min.

(b) tert-butyl(1R,3s,5S)-3-((4-chloro-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate(9′)

To a mixture of 2,4-dichloro-6-methoxypyrimidine (20 g, 112 mmol) and(1R,3s,5S)-tert-butyl3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (36.9 g, 145mmol) in THF (300 mL) was added DIPEA (39.0 mL, 223 mmol) and thereaction mixture was stirred at 20-25° C. overnight. Additional(1R,3s,5S)-tert-butyl3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (4.26 g, 16.76mmol) was added and the reaction mixture was warmed to 55° C., stirredfor 2.5 h, cooled to RT and stirred for 2 d. Heptane (400 mL) was addedover 30 min and the reaction mixture was filtered. The liquid phase wasazeotroped with IPA (300 mL, then 200 mL) to about 200-300 mL, stirredat 5° C. overnight, and filtered to give the title intermediate (29.2 g,71.4 mmol, 63.9% yield). HPLC Method A Retention time 28.27 min.

(c) tert-butyl(1R,3s,5S)-3-((4-methoxy-6-((5-(((triisopropylsilyl)oxy)methyl)-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate(7′)

To a 250 mL flask were added the product of the previous step (9′) (8 g,20.16 mmol), the product of step (a) (3′) (6.79 g, 25.2 mmol), Cs₂CO₃(13.13 g, 40.3 mmol), Xphos Pd G2 (0.793 g, 1.008 mmol) and XPhos (0.480g, 1.008 mmol), the reaction mixture was degassed three times and1,4-dioxane (80 mL) and water (8.00 mL) were added to give a suspension.The reaction mixture was degassed three times under vacuum and nitrogenand heated to 100° C., refluxed overnight, and cooled to 35° C.SiliaMetS® thiol functionalized silica (4 g) was added and the reactionmixture was warmed to 65° C., stirred for 2 h, cooled to 50° C. andfiltered through Celite® (5 g). Water (200 mL) and isopropyl acetate(200 mL) were added, the layers were separated, and the organic layerwas washed with 20% NaHSO₃. The layers were separated and the organiclayer was washed with brine. The layers were again separated and theaqueous layer was extracted with EtOAc (300 mL). Combined organic layerswere dried over Na₂SO₄ and evaporated to provide crude product (about 20g). Methanol (50 mL) was added and the reaction mixture was stirred atRT, filtered and washed with methanol (10 mL) to remove solid to givethe title compound as a methanol solution which was used directly in thenext step. HPLC Method A: Retention time 16.05 min.

(d)(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol(1)

To a 250 mL flask were added the crude product of the previous step (7′)in methanol (50 mL) (12.68 g, 20.136 mmol) and 3 M HCl in CPME (67.1 mL,201 mmol) and the reaction mixture was stirred at RT for 2 h, andfiltered to give the crude 3 HCl salt of the title compound (4.8 g, 9.94mmol, 49.4% yield).

To a flask was added the above crude 3 HCl salt (2 g, 4.14 mmol)followed by water (30 mL) and SiliaMetS thiol functionalized silica 40%w/w (0.8 g, 4.14 mmol) and the reaction mixture was warmed to 65° C.,stirred for 16 h, filtered through Celite and washed with water (1.5mL). Acetone (120 mL) was added and the reaction mixture was stirred atRT overnight, filtered, washed with acetone (10 mL) and dried in vacuoat 50° C. to provide the purified 3 HCl salt of title compound (1.05 g,2.175 mmol, 52.5% yield). HPLC Method A: Retention time 10.09 min.

(e)1-(((1R,3s,5S)-3-((4-((5-(Hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile

To a vial were added the product of the previous step (1) (0.83 g, 1.719mmol) and NMP (8.3 mL), followed by TEA (1.44 mL, 10.31 mmol). Thereaction mixture was stirred for 5-10 min. 3-Cyanoazetidine-1-sulfonylchloride (0.466 g, 2.58 mmol) was added at 22° C. After 2 h, water (25mL) was added over 30 min and the reaction mixture was stirred for 22 h.The reaction mixture was filtered and washed with water (2 mL) to givethe title product (0.9 g, 1.704 mmol, 99% yield) as a white solid. HPLCMethod A: Retention time 16.18 min.

Example 11: Crystalline1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileForm I

To a 100 mL flask was added1-(((1R,3s,5S)-3-((4-((3-(hydroxymethyl)-1H-pyrazol-5-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile(1 g, 1.932 mmol) and DMF (3.00 mL) followed by acetone (4 mL). Water (6mL) was then added over 5 min and the reaction mixture was stirredovernight and filtered, and the solid was washed with water and acetone,and dried for 30 min to provide the title compound (0.94 g, 1.816 mmol,94% yield). HPLC Method A: Retention time 16.30 min.

Example 12: Crystalline1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileForm I

To a 20 L flask were added(3-((2-(((1R,3s,5S)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol(1) (1.20 kg, 2.69 mol), NMP (4.8 kg) and TEA (1.36 kg, 13.45 mol) andthe reaction mixture was stirred at RT for 2 h. Next, NMP (2.4 kg) wasadded and the reaction mixture was stirred and cooled to 5-10° C. overabout 1 h. 3-Cyanoazetidine-1-sulfonyl chloride (0.58 kg, 3.23 mol) wasadded in three batches, added every 0.5 h and the reaction mixture wasstirred for 2 h and warmed to RT. Methanol (4.2 kg) was added, thereaction mixture was stirred for 0.5 h. Water (21.6 kg) was added over 3h and the reaction mixture was stirred for 0.5 h and filtered. The solidwas rinsed with methanol (1.0 kg) to give the crude title compound (1.33kg) of which 1.20 kg was dissolved in NMP (3.6 kg). Acetone (3.8 kg) wasadded, the solution was filtered and the filtrate was heated to 45-55°C. with stirring. Water (6.6 kg) was added over 6 h and the mixture wasstirred, cooled to 25-30° C. and filtered. The solid was rinsed with4:5.5 acetone:water (1.5 L) and dried to provide the title compound(1.21 kg, 99% purity, 85% yield) HPLC Method B: Retention time 15.23min. The product was micronized using an air jet milling process to thefollowing particle size distribution: X₁₀=0.70 μm, X₅₀=2.17 μm, andX₉₀=6.15 μm, where X_(n) is defined as the percentage of particlessmaller than n %.

Example 13: Crystalline1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileForm II

To a flask were added1-(((1R,3s,5S)-3-((4-((5-(Hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile(16 g, 30.9 mmol) and DMF (160 mL) and the reaction mixture wasfiltered. To the solution was added water (480 mL) over 30 min and thereaction mixture was warmed to 65° C., stirred overnight, cooled to RT,stirred for 20 h, and filtered. The solid was dried overnight to givethe title intermediate (11.8 g, 22.80 mmol, 73.8% yield). HPLC Method A:Retention time 16.19 min.

Examples 14-16: Properties of the Solid Forms of the Invention

Samples of the Form I and Form II crystalline freebase of1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileof Examples 12 and 13 respectively, were analyzed by powder X-raydiffraction (PXRD), differential scanning calorimetry (DSC),thermogravimetric analysis (TGA), and dynamic moisture sorption (DMS).

Example 14 Powder X-Ray Diffraction

The powder X-ray diffraction patterns of FIGS. 1 and 5 were obtainedwith a Bruker D8-Advance X-ray diffractometer using Cu-Kα radiation(λ=1.54051 Å) with output voltage of 45 kV and current of 40 mA. Theinstrument was operated in Bragg-Brentano geometry with incident,divergence, and scattering slits set to maximize the intensity at thesample. For measurement, a small amount of powder (5-25 mg) was gentlypressed onto a sample holder to form a smooth surface and subjected toX-ray exposure. The samples were scanned in 2θ-2θ mode from 2° to 35° in2θ with a step size of 0.02° and a scan speed of 0.30° seconds per step.The data acquisition was controlled by Bruker DiffracSuite measurementsoftware and analyzed by Jade software (version 7.5.1). The instrumentwas calibrated with a corundum standard, within ±0.02° two-theta angle.Observed PXRD 2θ peak positions and d-spacings are shown in Tables 4 and5, respectively for crystalline Form I and crystalline Form II. Thetwo-theta peak positions of the Form I micronized material listed inTable 4, were compared with peak positions of an unmicronized sampleprepared by the same synthetic process. The maximum difference observedin two-theta peak positions was 0.04 degrees.

TABLE 4 PXRD Data for Crystalline Form I 2-Theta d (Å) Area A % 8.899.94 460823 100 10.64 8.31 75783 16.4 10.99 8.05 116893 25.4 12.99 6.81153914 33.4 13.44 6.59 156394 33.9 14.54 6.09 66952 14.5 14.76 6.0085164 18.5 15.02 5.89 133090 28.9 15.74 5.63 59537 12.9 16.25 5.45 7549216.4 16.47 5.38 85726 18.6 20.16 4.40 443759 96.3 20.39 4.35 207159 45.020.93 4.24 66277 14.4 21.81 4.07 52296 11.3 22.22 4.00 79031 17.1 23.363.81 62926 13.7 23.56 3.77 92923 20.2 24.99 3.56 70165 15.2 25.27 3.52124788 27.1 26.25 3.39 98075 21.3 26.84 3.32 77303 16.8

TABLE 5 PXRD Data for the Crystalline Form II 2-Theta d (Å) Area A %8.87 9.96 342177 100 10.64 8.31 61376 17.9 11.01 8.03 86511 25.3 12.886.87 105843 30.9 13.47 6.57 107366 31.4 14.51 6.10 45973 13.4 14.72 6.0145739 13.4 15.04 5.88 77441 22.6 15.58 5.69 32094 9.4 16.51 5.36 4899614.3 16.88 5.25 18790 5.5 19.02 4.66 23117 6.8 20.00 4.44 190640 55.720.42 4.35 63067 18.4 20.73 4.28 56902 16.6 21.67 4.10 26834 7.8 22.303.98 47801 14 22.93 3.87 20999 6.1 23.32 3.81 40192 11.7 25.28 3.5257929 16.9 25.99 3.43 28870 8.4

Example 15: Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Analysissoftware. A sample of each crystalline form was accurately weighed intoa covered aluminum pan. After a 5 minute isothermal equilibration periodat 5° C., the sample was heated using a linear heating ramp of 10°C./min from 0° C. to 300° C. A representative DSC thermogram of the FormI and Form II crystalline freebase of the invention is shown in FIGS. 2and 6, respectively.

Thermogravimetric analysis (TGA) measurements were performed using a TAInstruments Model Q-50 module equipped with high resolution capability.Data were collected using TA Instruments Thermal Analyst controller andanalyzed using TA Instruments Universal Analysis software. A weighedsample was placed onto a platinum pan and scanned with a heating rate of10° C. from ambient temperature to 300° C. The balance and furnacechambers were purged with nitrogen flow during use. A representative TGAtrace of the Form I and Form II crystalline freebase of the invention isshown in FIGS. 3 and 7.

Example 16: Dynamic Moisture Sorption Assessment

Dynamic moisture sorption (DMS) measurement was performed using a VTIatmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, Fla.33016). A weighed sample was used and the humidity was lowest possiblevalue (close to 0% RH) at the start of the analysis. The DMS analysisconsisted of an initial drying step (0% RH) for 120 minutes, followed bytwo cycles of sorption and desorption with a scan rate of 5% RH/stepover the humidity range of 5% RH to 90% RH. The DMS run was performedisothermally at 25° C. A representative DMS trace for the Form I andForm II crystalline freebase of the invention is shown in FIGS. 4 and 8,respectively.

Biological Assays

The compounds of the invention have been characterized in one or more ofthe following biological assays.

Assay 1: Biochemical JAK and Off-Target Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compounds were pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pKi (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Test compounds having a higher pKi value in each of the four JAK assaysshow greater inhibition of JAK activity. Compounds of the inventiontested in this assay typically exhibited pKi values between about 7.5and about 10.3.

A panel of off-target tyrosine kinase assays (ABL1, Flt3, RET, FGFR2,NTRK1, and pDGFRβ) were developed using a similar methodology, withrecombinant enzymes obtained from Life Technologies and biotinylatedpeptide substrates synthesized at AnaSpec. All assays were carried outat ambient temperature with a final ATP concentration of 100 μM.Detection reagents, including Eu-anti-phosphotyrosine (pY20) antibodyand SureLight APC-SA, were purchased from Perkin Elmer. Emission ratiosignals (665 nm/615 nm) were recorded and utilized for data analysis,and the final results were expressed as pIC₅₀. Selected compounds testedin this assay typically exhibited pIC₅₀ values between about 5 and about6.5.

Assay 2: Cellular JAK Potency Assay: Inhibition of IL-13

The potency of test compounds for inhibition of the JAK-dependentcytokine interleuken-13 (IL-13) was assessed by measuring IL-13 (IL-13,R&D Systems) induced STAT6 phosphorylation in HT-29 human colorectaladenocarcinoma cells (ATCC).

The anti-STAT6 antibody (Cell Signaling Technologies) was conjugated toAlphaScreen acceptor beads (Perkin Elmer), while the anti-pSTAT6(pTyr641) antibody (Cell Signaling Technologies) was biotinylated usingEZ-Link Sulfo-NHS-Biotin (Thermo Scientific).

HT-29 cells were grown at 37° C. in a 5% CO₂ humidified incubator inMcCoy's 5a Modified medium (ATCC) supplemented with 10% FBS (Hyclone),100 U/mL penicillin, 100 μg/mL streptomycin (Life Technologies), and 2mM GlutaMAX (Life Technologies). On day 1 of the assay, cells wereseeded at a 7,500 cells/well density in white poly-D-lysine-coated384-well plates (Corning) with 25 μL medium, and were allowed to adhereovernight in the incubator. On day 2 of the assay, the medium wasremoved and replaced with 12 μL of assay buffer (Hank's Balanced SaltSolution/HBSS, 25 mM HEPES, and 1 mg/ml bovine serum albumin/BSA)containing dose-responses of test compounds. Compounds were seriallydiluted in DMSO and then diluted another 1000-fold in media to bring thefinal DMSO concentration to 0.1%. Cells were incubated with testcompounds at 37° C. for 1 h, and followed by the addition of 12 μL ofpre-warmed IL-13 (12 ng/ml in assay buffer) for stimulation. Afterincubating at 37° C. for 30 min, the assay buffer (containing compoundand IL-13) was removed, and 10 μL of cell lysis buffer (25 mM HEPES,0.1% SDS, 1% NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mM EGTA, and supplementwith Complete Ultra mini protease inhibitors and PhosSTOP from RocheDiagnostics). The plates were shaken at ambient temperature for 30 minbefore the addition of detection reagents. A mixture ofbiotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beads was addedfirst and incubated at ambient temperature for 2 h, followed by theaddition of streptavidin conjugated donor beads (Perkin Elmer). After aminimum of 2 h incubation, the assay plates were read on the EnVisionplate reader. AlphaScreen luminescence signals were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results wereexpressed as the negative logarithm of the IC₅₀ value, pIC₅₀.

Test compounds having a higher pIC₅₀ value in this assay show greaterinhibition of IL-13 induced STAT6 phosphorylation. Compounds of theinvention tested in this assay typically exhibited pIC₅₀ values betweenabout 6.0 and about 7.8.

Assay 3: JAK Cytotoxicity Assay

A CellTiter-Glo luminescent cell viability/cytotoxicity assay wascarried out in BEAS-2B human lung epithelial cells (ATCC) under thenormal growth condition.

Cells were grown at 37° C. in a 5% CO₂ humidified incubator in 50%DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 500 cells/well density in white 384-welltissue culture plates (Corning) with 25 μL medium, and were allowed toadhere overnight in the incubator. On day 2 of the assay, 5 μL of mediumcontaining dose-responses of test compounds was added, and incubated at37° C. for 48 h. 30 μL of CellTiter-Glo detection solution (Promega) wassubsequently added, mixed on an orbital shaker for 5 min, and incubatedfor additional 10 min before being read on the EnVision reader.Luminescence signals were recorded and percent DMSO control values werecalculated.

For dose-response analysis, percent DMSO control data were plotted vs.compound concentrations to derive dose-response curves by lineconnecting each data point. The concentration at which each curvecrosses the 15% inhibition threshold is defined as CC₁₅. Results wereexpressed as the negative logarithm of the CC₁₅ value, pCC₁₅.

It is expected that test compounds exhibiting a lower pCC₁₅ value inthis assay have less likelihood to cause cytotoxicity. Compounds of theinvention tested in this assay typically exhibited pCC₁₅ values betweenless than 5 and about 6.

In Vitro Assay Results

All of the compounds of Examples 1 to 9 and Tables 1 to 3 were tested inone or more of the assays described above. Since the JAK1 enzyme potencywas found and understood to be predictive of the cellular potencydescribed in Assay 2, enzyme characterization of certain compounds waslimited to the JAK1 enzyme.

In Table 6 below, for the JAK1, JAK 2, JAK3, and TYK2 enzyme assays, Arepresents a pK_(i) value≧10 (K_(i)≦0.1 nM), B represents a pK_(i) valuebetween 9 and 10 (K_(i) between 1 nM and 0.1 nM), C represents a pK_(i)value between 8 and 9 (K_(i) between 10 nM and 1 nM), and D represents apK_(i) value between 7.5 and 8 (K_(i) between 31.6 nM and 10 nM). Forthe THP-1 potency assay, A represents a pIC₅₀ value≧7.5 (IC₅₀≦32 nM), Brepresents a pIC₅₀ value between 6.7 and 7.5 (IC₅₀ between 200 nM and 32nM), and C represents a pIC₅₀ value between 6 and 6.7 (IC₅₀ between 1 μMand 200 nM).

TABLE 6 Example JAK 1 JAK 2 JAK 3 Tyk 2 HT-29 Number (pKi) (pKi) (pKi)(pKi) (pIC₅₀) 1 A A C B B 2 A A B B B 3 B A C B B 4 B A C B B 5 B B D CC 6 B 7 A A B B B 8 B B C B B 9 B B D B B Table 1 1-1 C B 1-2 B C 1-3 CC 1-4 D 1-5 B B 1-6 A B 1-7 B 1-8 A A 1-9 B B 1-10 B 1-11 B B 1-12 B B CB 1-13 A A B B A 1-14 B A C B A 1-15 B A C B B 1-16 B B C B B 1-17 B A CB A 1-18 B 1-19 B A Table 2 2-1 C C 2-2 B B 2-3 B B 2-4 B B 2-5 B B D C2-6 B B C B 2-7 B B 2-8 B B Table 3 3-1 B 3-2 B B B 3-3 B B C 3-4 B B B3-5 B B B 3-6 B B C 3-7 B B B 3-8 B B B

Assay 4: Cellular JAK Potency Assay: Inhibition of IL-4 StimulatedpSTAT6 in CD3+ T Cells

The potency of test compounds for inhibition of interleukin-4 (IL-4)stimulated STAT6 phosphorylation was measured in CD3-positive (CD3+) Tcells in human peripheral blood mononuclear cells (PBMCs) isolated fromhuman whole blood (Stanford Blood Center) using flow cytometry. BecauseIL-4 signals through JAK, this assay provides a measure of JAK cellularpotency.

CD3+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD3 antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences)was used to detect STAT6 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum(FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES(Life Technologies) and 1× Pen/Strep (Life Technologies). Cells wereseeded at 250,000 cells/well in media (200 μL), cultured for 1 h, andthen resuspended in assay media (50 μL) (RPMI supplemented with 0.1%bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1×Penstrep) containing various concentrations of test compounds. Compoundswere serially diluted in DMSO and then diluted another 500-fold (to a 2×final assay concentration) in assay media. Test compounds (50 μL) wereincubated with cells at 37° C., 5% CO₂ for 1 h, followed by the additionof 50 μL of IL-4 (R&D Systems; final concentration 20 ng/mL) inpre-warmed assay media for 30 min. After cytokine stimulation, cellswere fixed with pre-warmed fix solution (100 μL) (BD Biosciences) for 10min at 37° C., 5% CO₂, washed twice with FACS buffer (1 mL) (2% FBS inDPBS), and resuspended in 1000 μL of ice cold Perm Buffer III (BDBiosciences) for 30 min at 4° C. Cells were washed twice with FACSbuffer, and then resuspended in 100 μL of FACS buffer containinganti-CD3 PE (1:50 dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5dilution) for 60 min at room temperature in the dark. After incubation,cells were washed twice in FACS buffer before being analyzed using aLSRII flow cytometer (BD Biosciences).

To determine the inhibitory potency of test compounds in response toIL-4, the median fluorescent intensity (MFI) of pSTAT6 was measured inCD3+ T cells. IC₅₀ values were determined from analysis of theinhibition curves of MFI vs compound concentration. Data are expressedas pIC₅₀ (negative decadic logarithm IC₅₀) values (mean±standarddeviation). The compound of Example 2 exhibited a pIC₅₀ value of about7.3 in this assay.

Assay 5: Cellular JAK Potency Assay: Inhibition of IFNγ-Induced pSTAT1

The potency of test compounds for inhibition of interferon gamma (IFNγ)stimulated STAT1 phosphorylation was measured in CD14-positive (CD14+)monocytes derived from human whole blood (Stanford Blood Center) usingflow cytometry. Because IFNγ signals through JAK, this assay provides ameasure of JAK cellular potency.

Monocytes were identified using a fluorescein isothiocyanate (FITC)conjugated anti-CD14 antibody (Clone RM052, Beckman Coulter), and anAlexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, Clone 4a, BDBiosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Fetal Bovine Serum (FBS, LifeTechnologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (LifeTechnologies) and 1× Pen/Strep (Life Technologies). Cells were seeded at250,000 cells/well in media (200 μL), cultured for 2 h and resuspendedin assay media (50 μL) (RPMI supplemented with 0.1% bovine serum albumin(Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep) containing variousconcentrations of test compounds. Compounds were serially diluted inDMSO and then diluted another 1000-fold in media to bring the final DMSOconcentration to 0.1%. Test compounds were incubated with cells at 37°C., 5% CO₂ for 1 h, followed by the addition of pre-warmed IFNγ (R&DSystems) in media (50 μL) at a final concentration of 0.6 ng/mL for 30min. After cytokine stimulation, cells were fixed with pre-warmed fixsolution (100 μL) (BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with FACS buffer (1 mL) (1% BSA in PBS), resuspended in 1:10anti-CD14 FITC:FACS buffer (100 μL), and incubated at 4° C. for 15 min.Cells were washed once, and then resuspended in ice cold Perm Buffer III(BD Biosciences) (100 μL) for 30 min at 4° C. Cells were washed twicewith FACS buffer, and then resuspended in 1:10 anti-pSTAT1 Alexa Fluor647:FACS buffer (100 μL) for 30 min at RT in the dark, washed twice inFACS buffer, and analyzed using a LSRII flow cytometer (BD Biosciences).

To determine the inhibitory potency of test compounds, the medianfluorescent intensity (MFI) of pSTAT1 was measured in CD14+ monocytes.IC₅₀ values were determined from analysis of the inhibition curves ofMFI vs compound concentration. Data are expressed as pIC₅₀ (negativedecadic logarithm IC₅₀) values (mean±standard deviation). The compoundof Example 2 exhibited a pIC₅₀ value of about 7.6 in this assay.

Assay 6: Determination of Absorption in Cannulated Rats

Oral bioavailability (F %), fraction absorbed (F_(a) %) and fractionescaping hepatic clearance (F_(h) %) were determined in Sprague Dawleyrats from the following two studies:

(1) Pharmacokinetics in rats following an IV dose of test compound:Following IV dosing, plasma samples were typically collected from 0-6hr. Drug levels were determined using an LC-MS-MS method. The resultingdrug levels were used to compute the IV pharmacokinetic parameters: AUCIV and Dose IV.

(2) Rats that have been cannulated in their portal vein (PV) and also intheir jugular vein (JV) were dosed orally with test compound. Followingoral dosing, plasma samples were typically collected from 0-6 hr fromboth the portal vein and the jugular vein. Drug levels were determinedusing an LC-MS-MS method. The resulting drug levels were used to computethe following pharmacokinetic parameters: AUC PO PV, AUC PO JV, and DosePO.

Using data derived from the above studies, the oral bioavailability F %,and the quantities F_(a) % and F_(h) % were calculated from thefollowing formulas:

F %=(AUC PO JV/AUC IV)*(Dose IV/Dose PO)*100

Fa %=(AUC PO PV/AUC IV)*(Dose IV/Dose PO)*100

Fh %=AUC PO JV/AUC PO PV

where:

AUC PO JV=Area under the curve following oral dose and plasma collectedfrom the jugular vein

AUC PO PV=Area under the curve following oral dose and plasma collectedfrom the portal vein

AUC IV=Area under the curve following an intravenous dose

Dose IV=Intravenous Dose in mg/kg

Dose PO=Oral Dose in mg/kg

The compounds of Examples 1-4 were tested in this assay and exhibitedoral bioavailability (F %) less than about 25%. In particular, thecompounds of Examples 1, 2, and 4 exhibited F % values less than about5%. In addition, the compounds of Examples 1 and 2 exhibited absorptionat the portal vein (Fa %) less than about 25% while the compounds ofExamples 3 and 4 exhibited Fa % values greater than 40%.

Assay 7: Colon Pharmacokinetics in Rats

The test compound was formulated in 0.5% methyl-cellulose in water anddosed via oral gavage at 3.2 mg/kg and 100 mg/kg to Sprague Dawley rats.At various time points (typically 0.5, 1, 3, 6, 24 hr) post dosing,blood samples were removed via cardiac puncture and intact colons wereexcised from the rats. Blood samples were centrifuged at 1500×g for 15min to collect plasma. Colons were washed with ice cold phosphatebuffered saline (PBS), weighed, and homogenized at a dilution of 1:10 inPBS. Plasma and colon levels of test compound were determined by LC-MSanalysis against analytical standards constructed into a standard curvein the test matrix. A colon to plasma ratio was determined as the ratioof the colon AUC to the plasma AUC in μg hr/g. The compound of Example 2exhibited a colon to plasma ratio in excess of about 250 at 5 mg/kg andin excess of about 1200 at 100 mg/kg.

Assay 8: Mouse Model of Oxazalone-Induced Colitis

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis (Heller et al.Immunology, 2002, 17, 629-638). Adult BALB/C mice from Harlan were usedin the assay. On day 1, animals were lightly anesthetized withisoflurane and the hairs between the shoulder were carefully removedbefore oxazolone (4%, 150 μL, 4:1 acetone: olive oil formulation) orvehicle solution was slowly applied for skin sensitization. Seven daysafter skin sensitization, the mice were fasted overnight, anesthetizedwith isoflurane inhalation, and a 1 mL syringe equipped with a 3.5-Fcatheter, filled with oxazolone solution, was inserted carefully about 4cm into the colon of the mouse. Following insertion, 50 μL of theoxazolone solution (1%, 1:1 ethanol:water formulation) was injected veryslowly (over 30 sec using an injection pump) into the colon. Thecatheter was removed and the mice were held vertically (head down) for 2min to ensure that the entire oxazolone solution remained inside thecolon. Drug treatment (PO, BID or TID) or vehicle was initiated a dayprior to the oxazolone intrarectal (IR) challenge. Two-day postoxazolone intrarectal challenge, the Disease Activity Index (DAI) wasassessed by treatment-blinded experimenters for each mouse according tothe criteria score: stool consistency score (0, normal; 2, loose; 4,diarrhea), gross bleeding score (0, absence; 2, blood tinged; 4,presence), and weight loss score (0, none; 1, 1%-5%; 2, 5%-10%; 3,10%-20%; 4, more than 20%); DAI=average of (stool consistencyscore+gross bleeding score+weight loss score).

Selected compounds of the invention were tested in the assay. Efficacyin the model is evidenced by a decrease in DAI score as compared withthe score from vehicle treated animals. The compounds of examples 2 and4 exhibited a statistically significant decrease in DAI score ascompared with vehicle treated animals in the oxazalone model at a doseof 1, 3, and/or 10 mg/kg BID, while the compound of example 1 did notexhibit a statistically significant decrease at the doses up to 10 mg/kgBID tested in the assay.

Assay 9: Immunosuppression Effects in Mouse Splenic Natural Killer (NK)Cells

Depletion of mouse splenic cells is an experimental model ofimmunosuppression (Kudlacz et al., Am. J. of Transplantation, 2004, 4,51-57). The compound of Example 2 was assessed in the mouse splenic cellmodel following the same treatment paradigm as that used in theoxazolone-induced colitis model (Assay 8).

Adult male Balb/C mice (12-14 weeks of age) from Harlan were used forthe study. The compound (1, 10 and 100 mg/kg, BID) and tofacitinib (30and 60 mg/kg, BID) as a positive control were dosed orally for threedays to naïve mice. Spleens were harvested 1 h post last dose andcrushed immediately for cell subtype staining. Prior to fixation,fluorophore-labelled antibodies for CD19 (FITC; B cells), CD3e (PE; panT cells) and DX5 (APC; NK cells) were incubated with splenocyte samplesfrom each animal to allow for simultaneous, multiple subtype % analysison the flow cytometer. The number of total spleen cells for each animalwas measured by Scepter™ 2.0 Handheld Automated Cell Counter.

The absolute number of lymphocyte subtype population (e.g., splenic B, Tand NK cells) was calculated from the percentage of each subtype timestotal spleen cells for each animal. A one way ANOVA, with Dunnett's posthoc test, was used to compare the splenic lymphocytes number of thevehicle and test compound groups. The a level was set at p<0.05. Datawere presented as the mean±SEM for each group.

The positive control, tofacitinib (30 and 60 mg/kg; PO, BID),dose-dependently and significantly decreased splenic NK cell counts. Inthe same study, splenic NK cell counts were unaffected by the compoundof Example 2 at PO (BID) doses up to 100 mg/kg (the maximum dosetested). No treatment effect was observed for the B and T cellpopulations with either compound.

This data, in conjunction with the 1 mg/kg minimal dose that caused asignificant anti-colitic effect in the mouse model of oxazolone-inducedcolitis (Assay 8), allow a functional therapeutic index of >100 to becomputed for the compound of Example 2.

Assay 10: Dermal Pharmacokinetics in Mouse and Mini-Pig Skin

The objective of this assay was to determine the epidermal, dermal andplasma pharmacokinetics of a test compound following a 24 hr exposure tointact mouse or mini-pig skin.

The compound prepared in Example 12,1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,was formulated to 0.5% (w/w) in cream or ointment as described, asFormulation A or Formulation B, respectively in Table 7.

Twenty-four hours prior to dosing the hair was shaved from the back of25 g male Balb/c mice exposing an area at of least 6 cm² (about 10% ofbody surface) and, in a separate experiment, of 10 kg Gottingenmini-pigs exposing an area of at least 450 cm² (about 10% of bodysurface). At time zero, following isoflurane anesthesia, the testcompound was applied to the back of mice or mini-pigs at a dose of 25μL/cm². The skin was covered with an adhesive cover to prevent loss ofcompound to the cage or bedding.

Following 24 h exposure, the backs were gently washed with soap andwater to remove non-absorbed drug and patted dry. Immediately followingthis washing, blood was drawn by cardiac puncture from the mice and viavenipuncture from the mini-pigs. The outer skin (stratum corneum) wasthen removed by adhesive tape stripping. Upon exposure of the epidermisa 0.5 cm punch biopsy was taken. The epidermis and dermis were quicklyseparated, weighed and snap frozen. Similar samples were obtained at 48h post dosing in mice and at 48 h, 94 h, and 168 h (7 days) post-dosingin mini-pigs.

Epidermis and dermis samples were homogenized in 1:10 (w/v) water usinga Covaris ultrasonic homogenizer. Samples were extracted in 3 volumes ofacetonitrile and quantified against a standard curve via LC-MS analysis.As evidenced by the pharmacokinetic parameters AUC_(0-t). for plasma,epidermis and dermis shown in Table 8 below, significant compoundexposure was exhibited in epidermis and dermis layers while the plasmaexposure was negligible in mice and below the limit of quantitation inmini-pig.

TABLE 7 Formulation A Formulation B Compound of 0.5%   Compound of0.5%   Example 12 Example 12 Stearic Acid 5% Octylhydroxystearate 5%Cetostearyl Alcohol 5% C8-C10 Triglyceride 5% Isopropyl Palmitate 4%Vaseline (Petrolatum) 79.5%   Octylhydroxystearate 2%N-Methylpyrrolidone 10%  BRIJ S2 1.08%   (PEG 2 Stearyl Ether) BRIJ S206.92%   (PEG 20 Stearyl Ether) N-Methylpyrrolidine 10%  PEG400 10%  ROWater 55.5%  

TABLE 8 Plasma Epidermis Dermis AUC_(0-t) AUC_(0-t) AUC_(0-t) (μg *hr/mL) (μg * hr/g) (μg * hr/g) Mouse 0.014 718 61 Formulation A Mouse0.006 2830 296 Formulation B Mini-pig <0.001 988 71 Formulation AMini-pig <0.001 4030 114 Formulation B

Assay 11: Topical TPA-Induced Irritant Contact Dermatitis Model in Mice

The objective of this assay is to assess the anti-inflammatory effect ina model of acute dermatitis of test compounds being studied forcutaneous inflammatory conditions such as atopic dermatitis (Dong etal., J Pharmacol Exp Ther, 2013, 344, 436-446).

Topical dermal application of phorbol ester12-O-tetradecanoylphorbol-13-acetate (TPA) in mice causes aninflammatory response that is characterized by edema and neutrophilinflux at the early phase (2-24 h) and by epidermal cell proliferationat the later phase (24-48 h) (Griffiths et al., Agents and Actions,1988, 25, 344-351). Female Balb/c mice were topically administered witheither vehicle (1:7 DMSO:acetone) or 20 μL of a solution of TPA (2.5 μg)in vehicle on each ear. At 30 min before and 15 min after TPAadministration, either vehicle or the compound of Example 2 at doses of30, 100, 300, 1000, and 3000 μg in vehicle was applied topically to eachear. The degree of inflammation was assessed as the change in earthickness at 6 hours after TPA application. The compound of Example 2exhibited a dose- and concentration-dependent inhibition of TPA-inducedincrease in ear thickness. The maximum statistically significant effectwas 41% inhibition observed at the 1000 μg dose.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound of formula (I):

wherein R¹ is selected from: (a) —S(O)₂R⁴, wherein R⁴ is selected from:C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with —CN,—OC₁₋₃alkyl, or C₃₋₆cycloalkyl, heterocyclyl containing 4 to 6 ringatoms including one nitrogen atom, wherein any heterocyclyl isoptionally substituted with —CN, C₃₋₆cycloalkyl, pyridinyl, whereinpyridinyl is optionally substituted with fluoro, and phenyl; (b)C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with —CN,

or  pyridinyl, wherein pyridinyl is optionally substituted with —CN; and(c) —C(O)R⁵, wherein R⁵ is selected from: C₁₋₄alkyl, wherein C₁₋₄alkylis optionally substituted with C₃₋₆cycloalkyl, or with one or twofluoro, —OC₁₋₄alkyl, C₃₋₆cycloalkyl, and morpholinyl; R² is hydrogen ormethyl; R³ is C₁₋₃alkyl; and n is 1 or 2; or apharmaceutically-acceptable salt or stereoisomer thereof.
 2. Thecompound of claim 1, wherein R³ is methyl.
 3. The compound of claim 1,wherein R² is methyl.
 4. The compound of claim 1, wherein n is
 2. 5. Thecompound of claim 1, wherein R¹ is selected from: (a) S(O)₂R⁴, whereinR⁴ is selected from: C₁₋₂alkyl, wherein C₁₋₂alkyl is optionallysubstituted with —CN, —OCH₃, or cyclopropyl, azetidinyl or pyrrolidinyl,wherein azetidinyl is optionally substituted with —CN, cyclopropyl,pyridinyl, wherein pyridinyl is optionally substituted with fluoro, andphenyl; (b) C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with—CN,

or  pyridinyl, wherein pyridinyl is optionally substituted with —CN; and(c) C(O)R⁵, wherein R⁵ is selected from: C₁₋₂alkyl, wherein C₁₋₂alkyl isoptionally substituted with cyclopropyl, or with one or two fluoro,—OC₁₋₄alkyl, C₃₋₆cycloalkyl, and morpholinyl.
 6. The compound of claim1, wherein the compound is selected from:1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)sulfonyl)azetidine-3-carbonitrile,1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(methylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(((1R,3s,5S)-9-((2-methoxyethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,3-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-8-azabicyclo[3.2.1]octan-8-yl)propanenitrile,5-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,5-(((1R,3s,5S)-3-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)nicotinonitrile,isobutyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,2,2-difluoro-1-((1R,3s,5S)-3-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,(3-((2-(((1R,3s,5S)-9-(azetidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,1-(((1R,3s,5S)-3-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,(3-((2-(((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-(ethylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-((cyclopropylmethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,3-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)-sulfonyl)propanenitrile,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyrrolidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-(cyclopropylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-(azetidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-((cyclopropylmethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,4-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,(3-((6-methoxy-2-(((1R,3s,5S)-9-(pyridin-3-ylmethyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,3-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)propanenitrile,1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)cyclopropane-1-carbonitrile,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyridin-4-ylmethyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,4-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methyl)picolinonitrile,2,2-difluoro-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,isobutyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,methyl(1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate,((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxy-pyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)(morpholino)methanone,2-cyclopropyl-1-((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)ethan-1-one,cyclopentyl((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methanone,andcyclobutyl((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)methanone,and pharmaceutically acceptable salts thereof.
 7. The compound of claim1, wherein the compound is a compound of formula (II):

or a pharmaceutically-acceptable salt thereof.
 8. The compound of claim7, wherein R⁴ is methyl, ethyl, azetidinyl, pyrrolidinyl, cyclopropyl,pyridinyl, or phenyl, wherein ethyl is optionally substituted withmethoxy, azetidinyl is optionally substituted with —CN, and pyridinyl isoptionally substituted with fluoro.
 9. The compound of claim 7, whereinthe compound is selected from:1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(methylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(((1R,3s,5S)-9-((2-methoxyethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,3-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxy-pyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)-sulfonyl)propanenitrile,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyrrolidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-(cyclopropylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(pyridin-3-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((6-methoxy-2-(methyl((1R,3s,5S)-9-(phenylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-(azetidin-1-ylsulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,(3-((2-(((1R,3s,5S)-9-((cyclopropylmethyl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,and(3-((2-(((1R,3s,5S)-9-((5-fluoropyridin-3-yl)sulfonyl)-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)-6-methoxypyrimidin-4-yl)amino)-1H-pyrazol-5-yl)methanol,and pharmaceutically-acceptable salts thereof. 10.1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileof formula:

or a pharmaceutically-acceptable salt thereof. 11.1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile.12. A crystalline form of1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrile,wherein the crystalline form is characterized by a powder X-raydiffraction comprising diffraction peaks at 2θ values of 8.89±0.20,12.99±0.20, 13.44±0.20, and 20.16±0.20.
 13. The crystalline form ofclaim 12 wherein the powder X-ray diffraction pattern is furthercharacterized by having two or more additional diffraction peaks at 2θvalues selected from 10.64±0.20, 10.99±0.20, 15.02±0.20, 15.74±0.20,16.47±0.20, 20.93±0.20, 22.22±0.20, and 26.25±0.20.
 14. The crystallineform of claim 12, wherein the crystalline form is characterized by apowder X-ray diffraction pattern in which the peak positions aresubstantially in accordance with the peak positions of the pattern shownin FIG.
 1. 15. The crystalline form of claim 12 wherein the crystallineform is characterized by a differential scanning calorimetry tracerecorded at a heating rate of 10° C. per minute which shows a maximum inendothermic heat flow at a temperature between about 235° C. and about245° C.
 16. The crystalline form of claim 15, wherein the crystallineform is characterized by a differential scanning calorimetry tracesubstantially in accordance with that shown in FIG.
 2. 17. Apharmaceutical composition comprising a compound of any one of claims 1and 10 to 12 and a pharmaceutically-acceptable carrier.
 18. Thepharmaceutical composition of claim 17 further comprising one or moreother therapeutic agents useful for treating gastrointestinalinflammatory disease.
 19. A process for preparing a compound of formula(I):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R³, and nare as defined in claim 1, the process comprising: (a) reacting acompound of formula (III):

wherein R², R³, and n are as defined in claim 1, with (i) Cl—S(O)₂R⁴,wherein R⁴ is as defined in claim 1; (ii) a compound of formula L-R^(A)wherein L is a leaving group and R^(A) is C₁₋₄alkyl, wherein C₁₋₄alkylis optionally substituted with —CN,

or pyridinyl, wherein pyridinyl is optionally substituted with —CN; Cl—C(O)R⁵, wherein R⁵ is as defined in claim 1; or  (iv) HO—C(O)R⁵,wherein R⁵ is as defined as in claim 1; and (b) optionally forming apharmaceutically acceptable salt to provide a compound of formula (I) ora pharmaceutically acceptable salt thereof.
 20. A compound of formula(III):

wherein R², R³, and n are as defined in claim
 1. 21. The compound ofclaim 20, wherein R² and R³ are each methyl.
 22. A method of preparingthe crystalline form of1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrileas defined in claim 12, the method comprising: (a) dissolving1-(((1R,3s,5S)-3-((4-((5-(hydroxymethyl)-1H-pyrazol-3-yl)amino)-6-methoxypyrimidin-2-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonan-9-yl)sulfonyl)azetidine-3-carbonitrilein a diluent selected from N-methylpyrrolidone and dimethylformamide toform a reaction mixture; (b) adding acetone and water to the reactionmixture; and (c) isolating the crystalline form from the reactionmixture.
 23. A method of treating a gastrointestinal inflammatorydisease in a mammal the method comprising administering to the mammal acompound of any one of claims 1 and 10 to
 12. 24. The method of claim23, wherein the method further comprises administering one or more othertherapeutic agents useful for treating a gastrointestinal inflammatorydisease.
 25. The method of claim 23 wherein the gastrointestinalinflammatory disease is ulcerative colitis.
 26. A method of treating aninflammatory disease of the skin in a mammal, the method comprisingapplying a pharmaceutical composition comprising a compound of any oneof claims 1 and 10 to 12 to the skin of the mammal.
 27. The method ofclaim 26, wherein the inflammatory disease is atopic dermatitis.